Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Science.gov (United States)

Zakian, V A; Brewer, B J; Fangman, W L

1979-08-01

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA

Plasmids in Gram negatives: molecular typing of resistance plasmids.

Science.gov (United States)

Carattoli, Alessandra

2011-12-01

A plasmid is defined as a double stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under non-stressed conditions but they have systems which guarantee their autonomous replication also controlling the copy number and ensuring stable inheritance during cell division. Most of the plasmids confer positively selectable phenotypes by the presence of antimicrobial resistance genes. Plasmids evolve as an integral part of the bacterial genome, providing resistance genes that can be easily exchanged among bacteria of different origin and source by conjugation. A multidisciplinary approach is currently applied to study the acquisition and spread of antimicrobial resistance in clinically relevant bacterial pathogens and the established surveillance can be implemented by replicon typing of plasmids. Particular plasmid families are more frequently detected among Enterobacteriaceae and play a major role in the diffusion of specific resistance genes. For instance, IncFII, IncA/C, IncL/M, IncN and IncI1 plasmids carrying extended-spectrum beta-lactamase genes and acquired AmpC genes are currently considered to be "epidemic resistance plasmids", being worldwide detected in Enterobacteriaceae of different origin and sources. The recognition of successful plasmids is an essential first step to design intervention strategies preventing their spread. Copyright © 2011 Elsevier GmbH. All rights reserved.

Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F).

Science.gov (United States)

Ongkudon, Clarence M; Danquah, Michael K

2010-10-15

Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30-80°C), mobile phase flow rate (0.1-1.8mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50-80%), buffer pH (6-10), ionic strength of binding buffer (0.3-0.7M) and buffer gradient elution slope (1-10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400nm pore size of monolith in 0.7M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0M at 3%B/min. Copyright © 2010 Elsevier B.V. All rights reserved.

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 plasmids showed distinct properties: pEL5.7 was capable of replicating in L. casei MCJΔ1 and Lactobacillus delbrueckii subsp. lactic LBCH-1 but failed to do so in two other tested lactobacilli strains whereas pEL5.6 replicated in three different strains, including L. casei MCJΔ1, L. casei NJ, Lactobacillus......Many lactic acid bacteria carry different plasmids, particularly those that replicate via a theta mechanism. Here we describe Lactobacillus casei MCJ(CCTCC AB20130356), a new isolate that contains pMC11, carrying two distinct theta-type replicons. Each replicon contained an iteron in the origin...... of replication (oriV1 or oriV2) and a gene coding for the replicase (RepA_1 or RepB_1), both of which are essential for plasmid replication. Escherichia coli/Lactobacillus shuttle vectors were constructed with each replicon, yielding pEL5.7 and pEL5.6 that are based on oriV2 and oriV1 replicons, respectively...

Translational up-regulation and high-level protein expression from plasmid vectors by mTOR activation via different pathways in PC3 and 293T cells.

Directory of Open Access Journals (Sweden)

Prashanthi Karyala

Full Text Available BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B, β-galactosidase (β-gal and green fluorescent protein (GFP from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to

Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum

International Nuclear Information System (INIS)

Seetharam, S.; Protic-Sabljic, M.; Seidman, M.M.; Kraemer, K.H.

1987-01-01

A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C to A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation

Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line.

Science.gov (United States)

Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi

2012-01-01

TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting. The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

Bacteriophage T5 encodes a homolog of the eukaryotic transcription coactivator PC4 implicated in recombination-dependent DNA replication.

Science.gov (United States)

Steigemann, Birthe; Schulz, Annina; Werten, Sebastiaan

2013-11-15

The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors. © 2013.

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

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

Frequency and diversity of small cryptic plasmids in the genus Rahnella

Directory of Open Access Journals (Sweden)

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

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.

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 ...

Plasmids replicatable in Bacillus subtilis, E. coli and lactic acid streptococcus bacteria

NARCIS (Netherlands)

Kok, Jan; Maat, Jan; van der Vossen, Josephus Mauritius; Venema, Gerard

1997-01-01

The claimed invention is drawn to a recombinant plasmid which can replicate in Bacillus subtilis, Escherichia coli, and lactic acid Streptococcus bacteria comprising the replication of origin from Streptococcus cremoris plasmid pWV01 as its origin of replication, in addition to coding marker genes

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. Copyright © 2014 Elsevier Inc. All rights reserved.

The technology of large-scale pharmaceutical plasmid purification ...

African Journals Online (AJOL)

Further test demonstrated that the pcDNAlacZ purified with CTAB and authoritative endotoxin-free plasmid Kit had the similar transfection efficiency in vivo and in vitro. CTAB can be used for plasmid purification; the main advantages of the DNAs purified with CTAB include the avoidance of animal-derived enzymes, toxic ...

Initiation preference at a yeast origin of replication.

Science.gov (United States)

Brewer, B J; Fangman, W L

1994-04-12

Replication origins in the yeast Saccharomyces cerevisiae are identified as autonomous replication sequence (ARS) elements. To examine the effect of origin density on replication initiation, we have analyzed the replication of a plasmid that contains two copies of the same origin, ARS1. The activation of origins and the direction that replication forks move through flanking sequences can be physically determined by analyzing replication intermediates on two-dimensional agarose gels. We find that only one of the two identical ARSs on the plasmid initiates replication on any given plasmid molecule; that is, this close spacing of ARSs results in an apparent interference between the potential origins. Moreover, in the particular plasmid that we constructed, one of the two identical copies of ARS1 is used four times more frequently than the other one. These results show that the plasmid context is critical for determining the preferred origin. This origin preference is also exhibited when the tandem copies of ARS1 are introduced into a yeast chromosome. The sequences responsible for establishing the origin preference have been identified by deletion analysis and are found to reside in a portion of the yeast URA3 gene.

Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis

International Nuclear Information System (INIS)

Weinrauch, Y.; Dubnau, D.

1987-01-01

Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2 H and 15 N, in the presence of 32 Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases

Molecular processes as basis for plasmid-mediated bacterial UV-light resistance and mutagenesis

International Nuclear Information System (INIS)

Aleshkin, G.I.; Brukhanskij, G.V.; Skavronskaya, A.G.

1985-01-01

The increase of UV-resistance and UV-induced mutagenesis by lambda 1 pint intmid as well as molecular-genetic mechanisms of plasmid participation in reparation and DNA replication and its degradation after UV-irradiation in plasmid cells on pKM101 plasmid model have been investigated. Data testifying to the necessity of intmid integration in chromosome as obligatory stage of intmid participation in increasing UV-resistance of bacterial cells are obtained. It has been found that intmid raises UV-resistance of cells and increases respectively the UV-induced reverants efficiency. On the basis of the experiment data the conclusion is drawn that the intmid capacity to raise UV-resistance and, possibly, mutagenesis is bound not only with its integration into chromosome but also with pol A + chromosome replication by dependendent imtmid replication complex. It is shown that pKM101 plasmid ensures functioning in E coli cells of inducible, chloroamphenicol-resistant DNA replication, highly resistant to UV-light harmful effect and that the volume of excision reparation in E. coli cells carrying pKM101 plasmid is increased as compared with the volume of reparation in plasmid legs cells. The combination of the data obtained gives grounds to the authors to assume that inducible replication, inducible reparation of DNA and inducible decrease of DNA degradation determined by pKM101 plasmid may serve as recA + lexA + basis dependent increase of UV-resistance and mutagenesis and that these processes provide the possibility of functioning of integrative replication mechanism of plasmid participation in ensuring UV-resistance and mutagenesis of plants

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

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

International Nuclear Information System (INIS)

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 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 ® 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

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.

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

Directory of Open Access Journals (Sweden)

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.

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

OpenAIRE

Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

2008-01-01

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK h...

Herpesvirus papio contains a plasmid origin of replication that acts in cis interspecies with an Epstein-Barr virus trans-acting function.

Science.gov (United States)

Pesano, R L; Pagano, J S

1986-01-01

Herpesvirus papio (HVP) and Epstein-Barr virus (EBV) are closely related biologically and biochemically; lymphoblastoid cells infected with either virus contain episomal viral DNA. The putative origin of replication for EBV plasmids (oriP) has been assigned to a 1,790-base-pair fragment (cis) in the short unique region of the genome which requires a viral function supplied in trans from elsewhere in the genome (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We report here the identification of the putative origin of replication (cis) in HVP; we assigned it to the HVP EcoRI K fragment. The results indicate that the HVP replication process requires both a cis and a trans-acting function, analogous to that found in EBV. Images PMID:3023667

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

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

Science.gov (United States)

Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

2008-01-01

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli. PMID:18390685

Topology of a Membrane Associated Regulator of Prokaryotic DNA Replication

National Research Council Canada - National Science Library

Firshein, William

1998-01-01

This proposal has focused on a broad host range plasmid, RK2, as a model system to study how a pair of initiation proteins encoded by the plasmid for DNA replication function when replication occurs...

Comparative symbiotic plasmid analysis indicates that symbiosis gene ancestor type affects plasmid genetic evolution.

Science.gov (United States)

Wang, X; Zhao, L; Zhang, L; Wu, Y; Chou, M; Wei, G

2018-07-01

Rhizobial symbiotic plasmids play vital roles in mutualistic symbiosis with legume plants by executing the functions of nodulation and nitrogen fixation. To explore the gene composition and genetic constitution of rhizobial symbiotic plasmids, comparison analyses of 24 rhizobial symbiotic plasmids derived from four rhizobial genera was carried out. Results illustrated that rhizobial symbiotic plasmids had higher proportion of functional genes participating in amino acid transport and metabolism, replication; recombination and repair; carbohydrate transport and metabolism; energy production and conversion and transcription. Mesorhizobium amorphae CCNWGS0123 symbiotic plasmid - pM0123d had similar gene composition with pR899b and pSNGR234a. All symbiotic plasmids shared 13 orthologous genes, including five nod and eight nif/fix genes which participate in the rhizobia-legume symbiosis process. These plasmids contained nod genes from four ancestors and fix genes from six ancestors. The ancestral type of pM0123d nod genes was similar with that of Rhizobium etli plasmids, while the ancestral type of pM0123d fix genes was same as that of pM7653Rb. The phylogenetic trees constructed based on nodCIJ and fixABC displayed different topological structures mainly due to nodCIJ and fixABC ancestral type discordance. The study presents valuable insights into mosaic structures and the evolution of rhizobial symbiotic plasmids. This study compared 24 rhizobial symbiotic plasmids that included four genera and 11 species, illuminating the functional gene composition and symbiosis gene ancestor types of symbiotic plasmids from higher taxonomy. It provides valuable insights into mosaic structures and the evolution of symbiotic plasmids. © 2018 The Society for Applied Microbiology.

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

Characterization of Plasmid pPO1 from the Hyperacidophile Picrophilus oshimae

Directory of Open Access Journals (Sweden)

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.

The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family.

Science.gov (United States)

Li, Xiaobin; Top, Eva M; Wang, Yafei; Brown, Celeste J; Yao, Fei; Yang, Shan; Jiang, Yong; Li, Hui

2014-01-01

A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), 29 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T, and pTer331, suggesting these hypothetical orfs may represent "essential" plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world.

The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family

Directory of Open Access Journals (Sweden)

Xiaobin eLi

2015-01-01

Full Text Available A self-transmissible broad-host-range (BHR plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs, 28 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102 and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331, based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T and pTer331, suggesting these hypothetical orfs may represent ‘‘essential’’ plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world.

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.

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

2010-01-01

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 ΔasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain χ9447. 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/Pcro) (PR), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC PBAD 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 β-lactamase, and cloned into pYA4534 under the control of the Ptrc promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain χ9447 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. PMID:20308296

Regulation of DNA replication in irradiated cells by trans-acting factors

International Nuclear Information System (INIS)

Wang, Y.; Huq, M.S.; Cheng, X.; Iliakis, G.

1995-01-01

We compared DNA replication activity in cytoplasmic extracts prepared from irradiated and nonirradiated HeLa cells using a simian virus 40 (SV40)-based in vitro replication assay. The assay measures semi-conservative DNA replication in a plasmid carrying the SV40 origin of replication and requires SV40 T antigen as the sole noncellular protein. The plasmid DNA used in the replication reaction is never exposed to radiation. We find that replication of plasmid DNA is significantly reduced when cytoplasmic extracts from irradiated cells are used. Since plasmid replication proceeds to completion in extracts from irradiated cells, the observed reduction in the overall replication activity is probably due to a reduction in the efficiency of initiation events. The degree of inhibition of DNA replication after exposure to 10, 30 and 50 Gy X rays as measured in vitro using this assay is similar to that measured in intact cells immediately before processing for extract preparation. These observations are compatible with the induction or activation by ionizing radiation of a factor(s) that inhibits in trans DNA replication. The results contribute to our understanding of the mechanism(s) developed by the cells to regulate DNA replication when exposed to clastogenic agents. Such processes may be of significance in the restoration of DNA integrity, and may define yet another checkpoint operating during S at the level of clusters of replicons. 26 refs., 4 figs

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

Science.gov (United States)

Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

2012-01-01

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop + oriλ+ sequence. PMID:22590552

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

Science.gov (United States)

Inui, Masayuki; Roh, Jung Hyeob; Zahn, Kenneth; Yukawa, Hideaki

2000-01-01

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria. PMID:10618203

Construction and identification of eukaryotic expression vector of pcDNA3-UHRF1

International Nuclear Information System (INIS)

Li Xinli; Zhu Ran; Zhu Wei; Fan Saijun; Meng Qinghui

2011-01-01

Objective: To generate eukaryotic expression vector of pcDNA3-UHRF1(ubiquitin-like, containing PHD and RING finger domains 1, UHRF1) and testify its expression in breast cancer cells MDA-MB-231. Methods: A 2.3 kb cDNA fragment was amplified from the total RNA of the human breast cancer cells MCF-7 by the RT-PCR method and was cloned into the plasmid pcDNA3. The vector was identified by the double digestion with restriction enzymes Kpn I and Xho I and was sequenced. The cDNA of UHRF1 was transfected into human breast cancer cells MDA-MB-231 by Lipofactamin2000. The positive clones were selected by G418. The expression of the UHRF1 was detected by RT-PCR and Western blot analysis. Results: The recombinant eukaryotic expression vector pcDNA3-UHRF1 was digested with Kpn I and BamH I, and the electrophoresis of the digested products showed two fragments; 2.3kb fragment of UHRF1 and 5.4 kb fragment of pcDNA3, and the sequence inserted was identical to the published sequence. The MDA-MB-231 cells transfected with the pcDNA3-UHRF1 plasmid expressed a high level of the UHRF1 mRNA and protein. Conclusion: The recombinant eukaryotic cell expression vector of pcDNA3-UHRF1 is constructed successfully. The recombinant plasmid pcDNA3-UHRF1 can provide a very useful tool and lay an important foundation for the research on the function of UHRF1. (authors)

Genetic characterization of plasmid pRJ5 of Staphylococcus aureus compared to plasmid pE194

International Nuclear Information System (INIS)

Oliveira, S.S. de; Freire Bastos, M.C. de

1993-01-01

The pRJ5, a naturally occurring constitutive macrolide, lincosamide and streptogramin B (MLS) resistance plasmid of Staphylococcus aureus, was compared to pE194, a plasmid that confers the inducible phenotype. pRJ5 was stable in all strains of S. aureus tested, even under growth at 43 O C, which distinguished it from pE194 which was shown to be thermo-sensitive for replication. pRJ5, like pE194, was highly unstable in Bacillus subtilis when the cells were grown in nonselective conditions. Multimeric forms of pRJ5 DNA were detected in the few cells of B. subtilis that retained this plasmid. pE194 was transduced by phages φ 11 and φ 443 at frequencies 400 and 20-fold higher, respectively, than pRJ5. Both plasmids were co-transduced with the plasmid pRJ4. pRJ5 was shown to be compatible with pE194. Therefore they belong to distinct Inc groups. Hybridization studies revealed that pRJ5 shares a 1.35 kb region of homology to pE194, which is limited to the erm gene, conferring MLS resistance. (author)

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

Science.gov (United States)

Demarre, Gaëlle; Chattoraj, Dhruba K

2010-05-06

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.

The effect of dimethyl sulfoxide on the induction of DNA strand breaks in plasmid DNA and colony formation of PC Cl3 mammalian cells by alpha-, beta-, and Auger electron emitters (223)Ra, (188)Re, and (99m)Tc.

Science.gov (United States)

Runge, Roswitha; Oehme, Liane; Kotzerke, Jörg; Freudenberg, Robert

2016-12-01

DNA damage occurs as a consequence of both direct and indirect effects of ionizing radiation. The severity of DNA damage depends on the physical characteristics of the radiation quality, e.g., the linear energy transfer (LET). There are still contrary findings regarding direct or indirect interactions of high-LET emitters with DNA. Our aim is to determine DNA damage and the effect on cellular survival induced by (223)Ra compared to (188)Re and (99m)Tc modulated by the radical scavenger dimethyl sulfoxide (DMSO). Radioactive solutions of (223)Ra, (188)Re, or (99m)Tc were added to either plasmid DNA or to PC Cl3 cells in the absence or presence of DMSO. Following irradiation, single strand breaks (SSB) and double strand breaks (DSB) in plasmid DNA were analyzed by gel electrophoresis. To determine the radiosensitivity of the rat thyroid cell line (PC Cl3), survival curves were performed using the colony formation assay. Exposure to 120 Gy of (223)Ra, (188)Re, or (99m)Tc leads to maximal yields of SSB (80 %) in plasmid DNA. Irradiation with 540 Gy (223)Ra and 500 Gy (188)Re or (99m)Tc induced 40, 28, and 64 % linear plasmid conformations, respectively. DMSO prevented the SSB and DSB in a similar way for all radionuclides. However, with the α-emitter (223)Ra, a low level of DSB could not be prevented by DMSO. Irradiation of PC Cl3 cells with (223)Ra, (188)Re, and (99m)Tc pre-incubated with DMSO revealed enhanced survival fractions (SF) in comparison to treatment without DMSO. Protection factors (PF) were calculated using the fitted survival curves. These factors are 1.23 ± 0.04, 1.20 ± 0.19, and 1.34 ± 0.05 for (223)Ra, (188)Re, and (99m)Tc, respectively. For (223)Ra, as well as for (188)Re and (99m)Tc, dose-dependent radiation effects were found applicable for plasmid DNA and PC Cl3 cells. The radioprotection by DMSO was in the same range for high- and low-LET emitter. Overall, the results indicate the contribution of mainly indirect radiation

Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

Science.gov (United States)

Burbank, Lindsey P; Stenger, Drake C

2016-08-01

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

Conjugal properties of the Sinorhizobium meliloti plasmid mobilome.

Science.gov (United States)

Pistorio, Mariano; Giusti, María A; Del Papa, María F; Draghi, Walter O; Lozano, Mauricio J; Tejerizo, Gonzalo Torres; Lagares, Antonio

2008-09-01

The biology and biochemistry of plasmid transfer in soil bacteria is currently under active investigation because of its central role in prokaryote adaptation and evolution. In this work, we examined the conjugal properties of the cryptic plasmids present in a collection of the N(2)-fixing legume-symbiont Sinorhizobium meliloti. The study was performed on 65 S. meliloti isolates recovered from 25 humic soils of Argentina, which were grouped into 22 plasmid-profile types [i.e. plasmid operational taxonomic units (OTUs)]. The cumulative Shannon index calculated for the observed plasmid profiles showed a clear saturation plateau, thus indicating an adequate representation of the S. meliloti plasmid-profile types in the isolates studied. The results show that isolates of nearly 14% of the plasmid OTUs hosted transmissible plasmids and that isolates of 29% of the plasmid OTUs were able to retransfer the previously characterized mobilizable-cryptic plasmid pSmeLPU88b to a third recipient strain. It is noteworthy that isolates belonging to 14% of the plasmid OTUs proved to be refractory to the entrance of the model plasmid pSmeLPU88b, suggesting either the presence of surface exclusion phenomena or the occurrence of restriction incompatibility with the incoming replicon. Incompatibility for replication between resident plasmids and plasmid pSmeLPU88b was observed in c. 20% of the OTUs. The results reported here reveal a widespread compatibility among the conjugal functions of the cryptic plasmids in S. meliloti, and this fact, together with the observed high proportion of existing donor genotypes, points to the extrachromosomal compartment of the species as being an extremely active plasmid mobilome.

Presence and analysis of plasmids in human and animal associated Arcobacter species

DEFF Research Database (Denmark)

Douidah, Laid; De Zutter, Lieven; Van Nieuwerburgh, Filip

2014-01-01

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...

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.

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.

Isolation of a minireplicon of the plasmid pG6303 of Lactobacillus ...

Indian Academy of Sciences (India)

is a new mode of plasmid replication. [Fan J., Xi X., ... coli using the BioTeKe plasmid extraction kit (BioTeKe, Beijing, China) according .... media and incubated at 37◦C for three days. The methods of ..... Each experiment was repeated five times. Journal of ..... Cold Spring Harbor Laboratory Press, New York, USA. Soler N.

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

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.

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...

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of a 450-bp region of human papillomavirus type 1 that promotes episomal replication in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Chattopadhyay, Anasuya; Schmidt, Martin C.; Khan, Saleem A.

2005-01-01

Human papillomaviruses (HPVs) replicate as nuclear plasmids in infected cells. Since the DNA replication machinery is generally conserved between humans and Saccharomyces cerevisiae, we studied whether HPV-1 DNA can replicate in yeast. Plasmids containing a selectable marker (with or without a yeast centromere) and either the full-length HPV-1 genome or various regions of the viral long control region (LCR) and the 3' end of the L1 gene were introduced into S. cerevisiae and their ability to replicate episomally was investigated. Our results show that HPV-1 sequences promote episomal replication of plasmids although the yeast centromere is required for plasmid retention. We have mapped the autonomously replicating sequence activity of HPV-1 DNA to a 450 base-pair sequence (HPV-1 nt 6783-7232) that includes 293 nucleotides from the 5' region of the viral LCR and 157 nucleotides from the 3' end of the L1 gene. The HPV-1 ARS does not include the binding sites for the viral E1 and E2 proteins, and these proteins are dispensable for replication in S. cerevisiae

Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1

DEFF Research Database (Denmark)

Basta, Tamara; Smyth, John; Forterre, Patrick

2009-01-01

. 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...... in bacteria where relevant bacteriophages either are dependent on a conjugative plasmid for successful infection or are excluded by a resident plasmid....

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.

Activation of a yeast replication origin near a double-stranded DNA break.

Science.gov (United States)

Raghuraman, M K; Brewer, B J; Fangman, W L

1994-03-01

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.

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...

Construction of a recombinant eukaryotic human ZHX1 gene expression plasmid and the role of ZHX1 in hepatocellular carcinoma.

Science.gov (United States)

Wang, Jianping; Liu, Dejie; Liang, Xiaohong; Gao, Lifen; Yue, Xuetian; Yang, Yang; Ma, Chunhong; Liu, Jun

2013-11-01

The zinc-fingers and homeoboxes protein 1 (ZHX1) consists of 873 amino acid residues, is localized in the cell nucleus and appears to act as a transcriptional repressor. Previous studies have shown that ZHX1 interacts with nuclear factor Y subunit α (NF-YA), DNA methyltransferases (DNMT) 3B and ZHX2, all of which are involved in tumorigenesis. However, the exact role of ZHX1 in tumorigenesis remains unknown. The aim of the current study was to construct a recombinant eukaryotic expression plasmid containing the human ZHX1 (hZHX1) gene and to investigate the biological activities of ZHX1 in hepatocellular carcinoma (HCC). Reverse transcription-polymerase chain reaction (RT‑PCR) was used to amplify the N- and C-terminal fragments (ZHX1‑N and ZHX1‑C, respectively) of the hZHX1 gene. The two PCR fragments were cloned into the pEASY-T1 vector and subcloned into the pcDNA3 plasmid to generate a recombinant pcDNA3‑ZHX1 plasmid. Following identification by enzyme digestion and DNA sequencing, the recombinant pcDNA3‑ZHX1 plasmid was transfected into SMMC-7721 cells. The level of ZHX1 expression was detected by RT-PCR and western blot analysis. Cell growth curve assays were used to evaluate the effect of ZHX1 on cell proliferation. Moreover, the differential expression of ZHX1 between cancer and adjacent cirrhotic liver tissue was investigated by quantitative PCR (qPCR). Enzyme digestion and DNA sequencing confirmed the successful construction of the recombinant plasmid, pcDNA3‑ZHX1. qPCR and western blot analysis demonstrated that ZHX1 was efficiently expressed in SMMC-7721 cells and overexpression of ZHX1 may inhibit the proliferation of SMMC-7721 cells. In addition, reduced ZHX1 expression is widespread among cancer tissues from HCC patients. In conclusion, a recombinant eukaryotic expression plasmid, pcDNA3‑ZHX1, was successfully constructed. In addition, the current results indicate that a low expression of ZHX1 may be responsible for hepatocarcinogenesis.

On the 221 Rn → 221 Fr decay scheme

International Nuclear Information System (INIS)

Gromov, K.Ya.; Norseev, Yu.V.; Samatov, Zh.K.; Fominykh, V.I.; Chumin, V.G.; Kudrya, S.A.; Sergienko, V.A.

2002-01-01

The results of investigating the 221 Rnβ - - decay and the 225 Ac α-decay are compared. It is shown that 221 Fr levels at 145.9 and 393.2 keV are excited at the 221 Rn decay. Intensities and reduced probabilities of the β - - decay to the 221 Fr levels are determined. A conclusion is drawn that the parity of the 221 Rn ground state is positive

Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

DEFF Research Database (Denmark)

Reisner, A.; Molin, Søren; Zechner, E.L.

2002-01-01

An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination...... resistance genes and fluorescent markers. The choice of 5' non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E coli and is not restricted...

Mapping autonomously replicating sequence elements in a 73-kb ...

Indian Academy of Sciences (India)

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 ...

Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing.

Science.gov (United States)

Naito, Y; Naito, T; Kobayashi, I

1998-01-01

Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage. Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication. The number of transformants that appeared was far fewer than with the restriction-minus (r-) control. Most of the transformants were very small. After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control. Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid. These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid. Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature. This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection. This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+). A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.

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

Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid.

Science.gov (United States)

Zgaga, Z

1991-08-01

UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain. Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.

The qacC gene has recently spread between rolling circle plasmids of Staphylococcus, indicative of a novel gene transfer mechanism

Directory of Open Access Journals (Sweden)

Trudy M. Wassenaar

2016-09-01

Full Text Available Resistance of Staphylococcus species to quaternary ammonium compounds, frequently used as disinfectants and biocides, can be attributed to qac genes. These qac gene products belong to the Small Multidrug Resistant (SMR protein family, and are often encoded by rolling-circle (RC replicating plasmids. Four classes of SMR-type qac gene families have been described in Staphylococcus species: qacC, qacG, qacJ and qacH. Within their class, these genes are highly conserved, but qacC genes are extremely conserved, although they are found in variable plasmid backgrounds. The lower degree of sequence identity of these plasmids compared to the strict nucleotide conservation of their qacC means that this gene has recently spread. In the absence of insertion sequences or other genetic elements explaining the mobility, we sought for an explanation of mobilization by sequence comparison. Publically available sequences of qac genes, their flanking genes and the replication gene that is invariably present in RC-plasmids were compared to reconstruct the evolutionary history of these plasmids and to explain the recent spread of qacC. Here we propose a new model that explains how qacC is mobilized and transferred to acceptor RC-plasmids without assistance of other genes, by means of its location in between the Double Strand replication Origin (DSO and the Single-Strand replication Origin (SSO. The proposed mobilization model of this DSO-qacC-SSO element represents a novel mechanism of gene mobilization in RC-plasmids, which has also been employed by other genes, such as lnuA (conferring lincomycin resistance. The proposed gene mobility has aided to the wide spread of clinically relevant resistance genes in Staphylococcus populations.

Transfer of the lambdadv plasmid to new bacterial hosts

International Nuclear Information System (INIS)

Kellenberger-Gujer, G.; Boy de la Tour, E.; Berg, D.E.

1974-01-01

Lambda dv, which was derived from bacteriophage lambda, replicates autonomously as a plasmid in Escherichia coli and consists of only the immunity region (imm/sup lambda/) and DNA replication genes (O, P) of the ancestral phage. Addition phages (lambda imm 21 --lambda dv) carry the lambda dv fragment inserted as a tandem duplication in their genome (sequence A imm 21 O P imm/sup lambda/ O P R) are formed as recombinants after lambda imm 21 infection of strains carrying lambda dv. Addition phages were used to transfer lambda dv to new bacterial hosts. Lambda dv transfer by excision of the lambda dv segment from the addition phage genome requires a bacterial Rec or a phage Red recombination system. Successful transfer is stimulated by uv irradiation of the addition phage before infection. Some properties of the newly transferred lambda dv plasmids are described. (U.S.)

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.

Dynamics in copy numbers of five plasmids of a dairy Lactococcus lactis in dairy-related conditions including near-zero growth rates.

Science.gov (United States)

van Mastrigt, Oscar; Lommers, Marcel M A N; de Vries, Yorick C; Abee, Tjakko; Smid, Eddy J

2018-03-23

Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-encoded genes and the activity of the corresponding proteins is severely affected by changes in the number of plasmid copies. We studied the impact of growth rate on dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected which varied in size (3 to 39 kb), in replication mechanism (theta or rolling-circle) and in putative (dairy-associated) functions. Copy numbers ranged from 1.5 to 40.5 and the copy number of theta-type replicating plasmids were negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates showing that the plasmid replication rate was strictly controlled. One low-copy number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations reflected in complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation or presence of citrate (maximum 2.2-fold) signifying the stability in copy number of the plasmids. Importance Lactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation and oligopeptide uptake and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-encoded genes, it is important to know the factors that influence the plasmid copy numbers. We monitored plasmid copy numbers of L

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

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.

Plasmids of Selenomonas ruminantium and development of host-vector system

Czech Academy of Sciences Publication Activity Database

Hermanová, A.; Pristaš, P.; Molnárová, V.; Fliegerová, Kateřina; Javorský, P.

2001-01-01

Roč. 46, č. 4 (2001), s. 289-291 ISSN 0015-5632 Institutional research plan: CEZ:AV0Z5045916 Keywords : COMPLETE NUCLEOTIDE-SEQUENCE * CRYPTIC PLASMID * REPLICATION Subject RIV: EE - Microbiology, Virology Impact factor: 0.776, year: 2001

Replication and Transcription of Eukaryotic DNA in Esherichia coli

Science.gov (United States)

Morrow, John F.; Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Goodman, Howard M.; Helling, Robert B.

1974-01-01

Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA. Images PMID:4600264

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 initiating....... Furthermore, conjugation experiments were performed obtaining 30 transconjugants when selecting for antimicrobial resistance. Among them 19 gave no positive amplicons indicating presence of rep-families not tested for in this experimental setup....

Experimental study on anti-tumor effect of pcEgr-IFNγ gene-radiotherapy

International Nuclear Information System (INIS)

Wu Congmei; Li Xiuyi; Liu Shuzheng

2001-01-01

Objective: To study the anti-tumor effect of IFN γ gene-radiotherapy to murine melanoma and its immunologic mechanism. Methods: pcEgr-IFNγ plasmids were injected locally into tumor, and 36 hours later, the tumors were given 20 Gy X-ray irradiation. Tumor growth at different time, IFN γ expression 3 days later and immunologic indexes 15 days later were detected. Results: At 3-15 days after pcEgr-IFNγ gene-radiotherapy, the tumor growth rate was lower than that of irradiation alone group. It was also lower than that of gene therapy alone group and control plasmid combined with X-ray irradiation group significantly. Day 3 tumor IFN γ expression was higher than that of plasmid treatment alone group. NK activity, IL-2 and IFN γ secretion activities were higher than those of gene therapy alone and irradiation alone groups significantly. Conclusion: The antitumor effect of IFN γ gene-radiotherapy is better than that of either of them applied solely. Its mechanism might be concerned with the higher expression of IFN γ induced by irradiation in tumors and activation of anti-tumor immunologic functions

Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector▿

Science.gov (United States)

Lee, Ju-Hoon; Halgerson, Jamie S.; Kim, Jeong-Hwan; O'Sullivan, Daniel J.

2007-01-01

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three—a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria. PMID:17526779

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli

Directory of Open Access Journals (Sweden)

Lynn C. Thomason

2016-09-01

Full Text Available Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion.

A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

Science.gov (United States)

Rogers, Elizabeth E; Stenger, Drake C

2012-01-01

A ≈ 38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th) century.

A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

Directory of Open Access Journals (Sweden)

Elizabeth E Rogers

Full Text Available A ≈ 38kB plasmid (pXF-RIV5 was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51 sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th century.

Development and Host Compatibility of Plasmids for Two Important Ruminant Pathogens, Mycoplasma bovis and Mycoplasma agalactiae

Science.gov (United States)

Sharma, Shukriti; Citti, Chistine; Sagné, Eveline; Marenda, Marc S.

2015-01-01

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. PMID:25746296

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.

Plasmids from Food Lactic Acid Bacteria: Diversity, Similarity, and New Developments

Science.gov (United States)

Cui, Yanhua; Hu, Tong; Qu, Xiaojun; Zhang, Lanwei; Ding, Zhongqing; Dong, Aijun

2015-01-01

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. PMID:26068451

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.

Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

Science.gov (United States)

On, Kin Fan; Beuron, Fabienne; Frith, David; Snijders, Ambrosius P; Morris, Edward P; Diffley, John F X

2014-01-01

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins. PMID:24566989

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.

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....... The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact...

Transcriptome Sequence and Plasmid Copy Number Analysis of the Brewery Isolate Pediococcus claussenii ATCC BAA-344T during Growth in Beer

Science.gov (United States)

Pittet, Vanessa; Phister, Trevor G.; Ziola, Barry

2013-01-01

Growth of specific lactic acid bacteria in beer leads to spoiled product and economic loss for the brewing industry. Microbial growth is typically inhibited by the combined stresses found in beer (e.g., ethanol, hops, low pH, minimal nutrients); however, certain bacteria have adapted to grow in this harsh environment. Considering little is known about the mechanisms used by bacteria to grow in and spoil beer, transcriptome sequencing was performed on a variant of the beer-spoilage organism Pediococcus claussenii ATCC BAA-344T (Pc344-358). Illumina sequencing was used to compare the transcript levels in Pc344-358 growing mid-exponentially in beer to those in nutrient-rich MRS broth. Various operons demonstrated high gene expression in beer, several of which are involved in nutrient acquisition and overcoming the inhibitory effects of hop compounds. As well, genes functioning in cell membrane modification and biosynthesis demonstrated significantly higher transcript levels in Pc344-358 growing in beer. Three plasmids had the majority of their genes showing increased transcript levels in beer, whereas the two cryptic plasmids showed slightly decreased gene expression. Follow-up analysis of plasmid copy number in both growth environments revealed similar trends, where more copies of the three non-cryptic plasmids were found in Pc344-358 growing in beer. Transcriptome sequencing also enabled the addition of several genes to the P . claussenii ATCC BAA-344T genome annotation, some of which are putatively transcribed as non-coding RNAs. The sequencing results not only provide the first transcriptome description of a beer-spoilage organism while growing in beer, but they also highlight several targets for future exploration, including genes that may have a role in the general stress response of lactic acid bacteria. PMID:24040005

The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells.

Science.gov (United States)

Yen-Ting-Liu; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni

2014-10-01

The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications.

Inhibition of in vitro SV40 DNA replication by ultraviolet light

International Nuclear Information System (INIS)

Gough, G.; Wood, R.W.

1989-01-01

Ultraviolet light-induced DNA damage was found to inhibit SV40 origin-dependent DNA synthesis carried out by soluble humancell extracts. Replication of SV40-based plasmids was reduced to approx. 35% of that in unirradiated controls after irradiation with 50-100 J/m 2 germicidal ultraviolet light, where an average of 3-6 pyrimidine dimer photoproducts were formed per plasmid circle. Inhibition of the DNA helicase activity of T antigen (required for initiation of replication in the in vitro system) was also investigated, and was only significant after much higher fluences, 1000-5000 J/m 2 . The data indicate that DNA damage by ultraviolet light inhibits DNA synthesis in cell-free extracts principally by affecting components of the replication complex other than the DNA helicase activity of T antigen. The soluble system could be used to biochemically investigate the possible bypass or tolerance of DNA damage during replication (author). 21 refs.; 2 figs

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks.

Science.gov (United States)

Chen, Yuan-Jyue; Rao, Sundipta D; Seelig, Georg

2015-11-25

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Characterization of a Large Antibiotic Resistance Plasmid Found in Enteropathogenic Escherichia coli Strain B171 and Its Relatedness to Plasmids of Diverse E. coli and Shigella Strains.

Science.gov (United States)

Hazen, Tracy H; Michalski, Jane; Nagaraj, Sushma; Okeke, Iruka N; Rasko, David A

2017-09-01

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline ( tetA ), sulfonamides ( sulI ), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor ( csi ). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 ( csi and traI ) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli . Copyright © 2017 American Society for Microbiology.

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.

Isolation of a conjugative F-like plasmid from a multidrug-resistant Escherichia coli strain CM6 using tandem shock wave-mediated transformation.

Science.gov (United States)

Soto-Alonso, G; Cruz-Medina, J A; Caballero-Pérez, J; Arvizu-Hernández, I; Ávalos-Esparza, L M; Cruz-Hernández, A; Romero-Gómez, S; Rodríguez, A L; Pastrana-Martínez, X; Fernández, F; Loske, A M; Campos-Guillén, J

2015-07-01

Genetic characterization of plasmids from bacterial strains provides insight about multidrug resistance. Ten wild type Escherichia coli (E. coli) strains isolated from cow fecal samples were characterized by their antibiotic resistance profile, plasmid patterns and three different identification methods. From one of the strains, a fertility factor-like plasmid was replicated using tandem shock wave-mediated transformation. Underwater shock waves with a positive pressure peak of up to approximately 40 MPa, followed by a pressure trough of approximately -19 MPa were generated using an experimental piezoelectric shock wave source. Three different shock wave energies and a fixed delay of 750 μs were used to study the relationship between energy and transformation efficiency (TE), as well as the influence of shock wave energy on the integrity of the plasmid. Our results showed that the mean shock wave-mediated TE and the integrity of the large plasmid (~70 kb) were reduced significantly at the energy levels tested. The sequencing analysis of the plasmid revealed a high identity to the pHK17a plasmid, including the replication system, which was similar to the plasmid incompatibility group FII. It also showed that it carried an extended spectrum beta-lactamase gene, ctx-m-14. Furthermore, diverse genes for the conjugative mechanism were identified. Our results may be helpful in improving methodologies for conjugative plasmid transfer and directly selecting the most interesting plasmids from environmental samples. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Bacteria with multiple chromosomes represent up to 10% of all bacterial species. Unlike eukaryotes, these bacteria use chromosome-specific initiators for their replication. In all cases investigated, the machineries for secondary chromosome replication initiation are of plasmid origin. One of the...

Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites

Science.gov (United States)

Mortusewicz, Oliver; Roth, Wera; Li, Na; Cardoso, M. Cristina; Meisterernst, Michael; Leonhardt, Heinrich

2008-01-01

The multifunctional nuclear protein positive cofactor 4 (PC4) is involved in various cellular processes including transcription, replication, and chromatin organization. Recently, PC4 has been identified as a suppressor of oxidative mutagenesis in Escherichia coli and Saccharomyces cerevisiae. To investigate a potential role of PC4 in mammalian DNA repair, we used a combination of live cell microscopy, microirradiation, and fluorescence recovery after photobleaching analysis. We found a clear accumulation of endogenous PC4 at DNA damage sites introduced by either chemical agents or laser microirradiation. Using fluorescent fusion proteins and specific mutants, we demonstrated that the rapid recruitment of PC4 to laser-induced DNA damage sites is independent of poly(ADP-ribosyl)ation and γH2AX but depends on its single strand binding capacity. Furthermore, PC4 showed a high turnover at DNA damages sites compared with the repair factors replication protein A and proliferating cell nuclear antigen. We propose that PC4 plays a role in the early response to DNA damage by recognizing single-stranded DNA and may thus initiate or facilitate the subsequent steps of DNA repair. PMID:19047459

Brownian Ratchet Mechanism for Faithful Segregation of Low-Copy-Number Plasmids.

Science.gov (United States)

Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi; Neuman, Keir C; Liu, Jian

2017-04-11

Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division. In contrast, most low-copy-number plasmids need to be actively partitioned by a conserved tripartite ParA-type system. ParA is an ATPase that binds to chromosomal DNA; ParB is the stimulator of the ParA ATPase and specifically binds to the plasmid at a centromere-like site, parS. ParB stimulation of the ParA ATPase releases ParA from the bacterial chromosome, after which it takes a long time to reset its DNA-binding affinity. We previously demonstrated in vitro that the ParA system can exploit this biochemical asymmetry for directed cargo transport. Multiple ParA-ParB bonds can bridge a parS-coated cargo to a DNA carpet, and they can work collectively as a Brownian ratchet that directs persistent cargo movement with a ParA-depletion zone trailing behind. By extending this model, we suggest that a similar Brownian ratchet mechanism recapitulates the full range of actively segregated plasmid motilities observed in vivo. We demonstrate that plasmid motility is tuned as the replenishment rate of the ParA-depletion zone progressively increases relative to the cargo speed, evolving from diffusion to pole-to-pole oscillation, local excursions, and, finally, immobility. When the plasmid replicates, the daughters largely display motilities similar to that of their mother, except that when the single-focus progenitor is locally excursive, the daughter foci undergo directed segregation. We show that directed segregation maximizes the fidelity of plasmid partition. Given that local excursion and directed segregation are the most commonly observed modes of plasmid motility in vivo, we suggest that the operation of the ParA-type partition system has been shaped by evolution for high fidelity of plasmid segregation

Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector.

Directory of Open Access Journals (Sweden)

Yibing Wang

2011-09-01

Full Text Available Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB. Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP. As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome

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.

Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

Science.gov (United States)

Rhee, Mun Su; Kim, Jin-Woo; Qian, Yilei; Ingram, L O; Shanmugam, K T

2007-07-01

Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

Plasmids and rickettsial evolution: insight from Rickettsia felis.

Directory of Open Access Journals (Sweden)

Joseph J Gillespie

2007-03-01

Full Text Available 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.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.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 the acquirement of virulence traits in pathogenic strains, and the

The cost of copy number in a selfish genetic element: the 2-μm plasmid of Saccharomyces cerevisiae.

Science.gov (United States)

Harrison, Ellie; Koufopanou, V; Burt, A; MacLean, R C

2012-11-01

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade-off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1.

Science.gov (United States)

Galardi, Silvia; Mercatelli, Neri; Giorda, Ezio; Massalini, Simone; Frajese, Giovanni Vanni; Ciafrè, Silvia Anna; Farace, Maria Giulia

2007-08-10

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27(Kip1). We recognize two target sites for the microRNAs in the 3' untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G(1) to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27(Kip1) in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) down-regulation.

Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids.

Science.gov (United States)

He, Susu; Chandler, Michael; Varani, Alessandro M; Hickman, Alison B; Dekker, John P; Dyda, Fred

2016-12-06

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. The spread of antibiotic resistance among Gram-negative bacteria is a serious public health threat, as it can critically limit the types of drugs that can be used to treat infected patients. In particular, carbapenem-resistant members of the Enterobacteriaceae family are responsible for a significant and growing burden of morbidity and mortality. Here, we report on the mechanisms underlying the evolution of several plasmids carried by previously sequenced clinical Enterobacteriaceae isolates from the National Institutes of Health Clinical Center (NIH CC). Our ability to track genetic rearrangements that occurred within resistance plasmids was dependent on accurate annotation of the mobile genetic elements within the plasmids, which was greatly aided by access to long-read DNA sequencing data and knowledge of their mechanisms. Mobile genetic elements such as

49 CFR 571.221 - Standard No. 221, School bus body joint strength.

Science.gov (United States)

2010-10-01

... 49 Transportation 6 2010-10-01 2010-10-01 false Standard No. 221, School bus body joint strength. 571.221 Section 571.221 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION FEDERAL MOTOR VEHICLE SAFETY STANDARDS Federal Motor Vehicle Safety Standard...

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

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...

Multiple determinants controlling activation of yeast replication origins late in S phase.

Science.gov (United States)

Friedman, K L; Diller, J D; Ferguson, B M; Nyland, S V; Brewer, B J; Fangman, W L

1996-07-01

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.

RNase H and replication of ColE1 DNA in Escherichia coli.

OpenAIRE

Naito, S; Uchida, H

1986-01-01

Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase ...

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.

PC_Eyewitness and the sequential superiority effect: computer-based lineup administration.

Science.gov (United States)

MacLin, Otto H; Zimmerman, Laura A; Malpass, Roy S

2005-06-01

Computer technology has become an increasingly important tool for conducting eyewitness identifications. In the area of lineup identifications, computerized administration offers several advantages for researchers and law enforcement. PC_Eyewitness is designed specifically to administer lineups. To assess this new lineup technology, two studies were conducted in order to replicate the results of previous studies comparing simultaneous and sequential lineups. One hundred twenty university students participated in each experiment. Experiment 1 used traditional paper-and-pencil lineup administration methods to compare simultaneous to sequential lineups. Experiment 2 used PC_Eyewitness to administer simultaneous and sequential lineups. The results of these studies were compared to the meta-analytic results reported by N. Steblay, J. Dysart, S. Fulero, and R. C. L. Lindsay (2001). No differences were found between paper-and-pencil and PC_Eyewitness lineup administration methods. The core findings of the N. Steblay et al. (2001) meta-analysis were replicated by both administration procedures. These results show that computerized lineup administration using PC_Eyewitness is an effective means for gathering eyewitness identification data.

Antibacterial effect of cationic porphyrazines and anionic phthalocyanine and their interaction with plasmid DNA

Science.gov (United States)

Hassani, Leila; Hakimian, Fatemeh; Safaei, Elham; Fazeli, Zahra

2013-11-01

Resistance to antibiotics is a public health issue and identification of new antibacterial agents is one of the most important goals of pharmacological research. Among the novel developed antibacterial agents, porphyrin complexes and their derivatives are ideal candidates for use in medical applications. Phthalocyanines differ from porphyrins by having nitrogen atoms link the individual pyrrol units. The aza analogues of the phthalocyanines (azaPcs) such as tetramethylmetalloporphyrazines are heterocyclic Pc analogues. In this investigation, interaction of an anionic phthalocyanine (Cu(PcTs)) and two cationic tetrapyridinoporphyrazines including [Cu(2,3-tmtppa)]4+ and [Cu(3,4-tmtppa)]4+ complexes with plasmid DNA was studied using spectroscopic and gel electrophoresis methods. In addition, antibacterial effect of the complexes against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria was investigated using dilution test method. The results indicated that both porphyrazines have significant antibacterial properties, but Cu(PcTs) has weak antibacterial effect. Compairing the binding of the phthalocyanine and the porphyrazines to DNA demonstrated that the interaction of cationic porphyrazines is stronger than the anionic phthalocyanine remarkably. The extent of hypochromicity and red shift of absorption spectra indicated preferential intercalation of the two porphyrazine into the base pairs of DNA helix. Gel electrophoresis result implied Cu(2,3-tmtppa) and Cu(3,4-tmtppa) are able to perform cleavage of the plasmid DNA. Consequently, DNA binding and cleavage might be one of the antibacterial mechanisms of the complexes.

Effective inhibition of foot-and-mouth disease virus (FMDV replication in vitro by vector-delivered microRNAs targeting the 3D gene

Directory of Open Access Journals (Sweden)

Cai Xuepeng

2011-06-01

Full Text Available Abstract Background Foot-and-mouth disease virus (FMDV causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined. Results Four pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV. Conclusion Our results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.

Initiation at closely spaced replication origins in a yeast chromosome.

Science.gov (United States)

Brewer, B J; Fangman, W L

1993-12-10

Replication of eukaryotic chromosomes involves initiation at origins spaced an average of 50 to 100 kilobase pairs. In yeast, potential origins can be recognized as autonomous replication sequences (ARSs) that allow maintenance of plasmids. However, there are more ARS elements than active chromosomal origins. The possibility was examined that close spacing of ARSs can lead to inactive origins. Two ARSs located 6.5 kilobase pairs apart can indeed interfere with each other. Replication is initiated from one or the other ARS with equal probability, but rarely (< 5%) from both ARSs on the same DNA molecule.

The qacC Gene Has Recently Spread between Rolling Circle Plasmids of Staphylococcus, Indicative of a Novel Gene Transfer Mechanism

DEFF Research Database (Denmark)

Wassenaar, Trudy M; Ussery, David W; Ingmer, Hanne

2016-01-01

and transferred to acceptor RC-plasmids without assistance of other genes, by means of its location in between the Double Strand replication Origin (DSO) and the Single-Strand replication Origin (SSO). The proposed mobilization model of this DSO-qacC-SSO element represents a novel mechanism of gene mobilization...

Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Directory of Open Access Journals (Sweden)

Laurel D Wright

2015-10-01

Full Text Available We identified a functional single strand origin of replication (sso in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA. In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1 maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2 stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso's in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Adenoviral DNA replication: DNA sequences and enzymes required for initiation in vitro

International Nuclear Information System (INIS)

Stillman, B.W.; Tamanoi, F.

1983-01-01

In this paper evidence is provided that the 140,000-dalton DNA polymerase is encoded by the adenoviral genome and is required for the initiation of DNA replication in vitro. The DNA sequences in the template DNA that are required for the initiation of replication have also been identified, using both plasmid DNAs and synthetic oligodeoxyribonucleotides. 48 references, 7 figures, 1 table

Comparative genomics of the pIPO2/pSB102 family of environmental plasmids : sequence, evolution, and ecology of pTer331 isolated from Collimonas fungivorans Ter331

NARCIS (Netherlands)

Mela, Francesca; Fritsche, Kathrin; Boersma, Hidde; van Elsas, Jan D.; Bartels, Daniela; Meyer, Folker; de Boer, Wietse; van Veen, Johannes A.; Leveau, Johan H. J.

2008-01-01

Plasmid pTer331 from the bacterium Collimonas fungivorans Ter331 is a new member of the pIPO2/pSB102 family of environmental plasmids. The 40 457-bp sequence of pTer331 codes for 44 putative ORFs, most of which represent genes involved in replication, partitioning and transfer of the plasmid. We

Plasmid segregation mechanisms

DEFF Research Database (Denmark)

Ebersbach, G.; Gerdes, Kenn

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...... that segregate plasmids paired at mid-cell to daughter cells. Like microtubules, ParM filaments exhibit dynamic instability (i.e., catastrophic decay) whose regulation is an important component of the DNA segregation process. The Walker box ParA ATPases are related to MinD and form highly dynamic, oscillating...... filaments that are required for the subcellular movement and positioning of plasmids. The role of the observed ATPase oscillation is not yet understood. However, we propose a simple model that couples plasmid segregation to ParA oscillation. The model is consistent with the observed movement...

Elimination of indigenous linear plasmids in Streptomyces hygroscopicus var. jinggangensis and Streptomyces sp. FR008 to increase validamycin A and candicidin productivities.

Science.gov (United States)

Lu, Chenyang; Wu, Hang; Su, Xiurong; Bai, Linquan

2017-05-01

Giant linear plasmids, which replicate independently of the chromosomes, widely exist in actinobacteria. Previous studies mostly focused on the replication and evolution of the linear plasmids or the secondary metabolite gene clusters and the resistance gene clusters therein. However, the relationships of the linear plasmids to the productivities of secondary metabolites have not been studied. In this work, we developed a method to eliminate the indigenous linear plasmid pSHJG1 in Streptomyces hygroscopicus var. jinggangensis, and validamycin A titer increased by 12.5% (from 19.16 ± 1.93 to 21.56 ± 2.25 g/L) in the high-yielding strain TL01 and 43.7% (from 4.67 ± 0.05 to 6.71 ± 0.21 g/L) in the wild-type strain 5008, whereas the cellular growth of the plasmid-cured mutant was reduced. Subsequently, the plasmid-cured mutant was complemented with three structure genes involved in cellular growth in pSHJG1 under the control of a strong PvalA promoter. Among them, the complementation of genes pSHJG1.069 and pSHJG1.072, encoding a putative hydrolase and putative P-loop ATPase, respectively, resulted in the restoration of cellular growth and validamycin A titer. Furthermore, the elimination of indigenous linear plasmid pHZ228 in the candicidin producer Streptomyces sp. FR008 also led to enhanced candicidin production and reduced cellular growth. Because of the wide distribution of indigenous linear plasmids in actinobacteria, the engineering strategy described here could be implemented in a variety of strains for the overproduction of various natural products.

50 CFR 665.221 - Definitions.

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Definitions. 665.221 Section 665.221..., DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Hawaii Fisheries § 665.221 Definitions...) Man grey reef shark Carcharhinus amblyrhynchos. Man galapagos shark Carcharhinus galapagensis. Man...

Nucleotide Sequence and Characterization of the Broad-Host-Range Lactococcal Plasmid pWVO1

NARCIS (Netherlands)

Leenhouts, Cornelis; Tolner, Berend; Bron, Sierd; Kok, Jan; Venema, Gerhardus; Seegers, Jozef

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for

Analysis of cis-elements that facilitate extrachromosomal persistence of human papillomavirus genomes

International Nuclear Information System (INIS)

Pittayakhajonwut, Daraporn; Angeletti, Peter C.

2008-01-01

Human papillomaviruses (HPVs) are maintained latently in dividing epithelial cells as nuclear plasmids. Two virally encoded proteins, E1, a helicase, and E2, a transcription factor, are important players in replication and stable plasmid maintenance in host cells. Recent experiments in yeast have demonstrated that viral genomes retain replication and maintenance function independently of E1 and E2 [Angeletti, P.C., Kim, K., Fernandes, F.J., and Lambert, P.F. (2002). Stable replication of papillomavirus genomes in Saccharomyces cerevisiae. J. Virol. 76(7), 3350-8; Kim, K., Angeletti, P.C., Hassebroek, E.C., and Lambert, P.F. (2005). Identification of cis-acting elements that mediate the replication and maintenance of human papillomavirus type 16 genomes in Saccharomyces cerevisiae. J. Virol. 79(10), 5933-42]. Flow cytometry studies of EGFP-reporter vectors containing subgenomic HPV fragments with or without a human ARS (hARS), revealed that six fragments located in E6-E7, E1-E2, L1, and L2 regions showed a capacity for plasmid stabilization in the absence of E1 and E2 proteins. Interestingly, four fragments within E7, the 3' end of L2, and the 5' end of L1 exhibited stability in plasmids that lacked an hARS, indicating that they possess both replication and maintenance functions. Two fragments lying in E1-E2 and the 3' region of L1 were stable only in the presence of hARS, that they contained only maintenance function. Mutational analyses of HPV16-GFP reporter constructs provided evidence that genomes lacking E1 and E2 could replicate to an extent similar to wild type HPV16. Together these results support the concept that cellular factors influence HPV replication and maintenance, independently, and perhaps in conjunction with E1 and E2, suggesting a role in the persistent phase of the viral lifecycle

49 CFR 107.221 - Determination.

Science.gov (United States)

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Determination. 107.221 Section 107.221... PROGRAM PROCEDURES Preemption Waiver of Preemption Determinations § 107.221 Determination. (a) After... Chief Counsel issues a determination. (b) The Chief Counsel may issue a waiver of preemption only on...

Dicty_cDB: VHK221 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available VH (Link to library) VHK221 (Link to dictyBase) - - - - VHK221P (Link to Original site) VHK221F 603 VHK2...21Z 724 VHK221P 1307 - - Show VHK221 Library VH (Link to library) Clone ID VHK221 (Link... to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/VH/VHK2...-A/VHK221Q.Seq.d/ Representative seq. ID VHK221P (Link to... Original site) Representative DNA sequence >VHK221 (VHK221Q) /CSM/VH/VHK2-A/VHK221Q.Seq.d/ AACTCTCGAGTGCAAA

Dicty_cDB: SLH221 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available SL (Link to library) SLH221 (Link to dictyBase) - - - Contig-U16381-1 SLH221Z (Link... to Original site) - - SLH221Z 595 - - - - Show SLH221 Library SL (Link to library) Clone ID SLH221 (Link to...ycdb.biol.tsukuba.ac.jp/CSM/SL/SLH2-A/SLH221Q.Seq.d/ Representative seq. ID SLH22...1Z (Link to Original site) Representative DNA sequence >SLH221 (SLH221Q) /CSM/SL/SLH2-A/SLH221Q.Seq.d/ XXXXX...LH722 (SLH722Q) /CSM/SL/SLH7-A/SLH722Q.Seq.d/ 559 e-158 SLH221 (SLH221Q) /CSM/SL/SLH2-A/SLH221Q.Seq.d/ 559 e

Nucleotide sequence of pOLA52: a conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux

DEFF Research Database (Denmark)

Norman, Anders; Hansen, Lars H.; She, Qunxin

2008-01-01

. The plasmid was also classified as IncX1 with incompatibility testing. The conjugal transfer and plasmid maintenance regions of pOLA52 therefore seem to represent IncX1 orthologues of the well-characterized IncX2 plasmid R6K. Sequence homology searches in GenBank also suggested a considerably higher...... of type 3 fimbriae (mrkABCDF). The plasmid was found to be 51,602 bp long with 68 putative genes. About half of the plasmid constituted a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin (TA) plasmid addiction system...... prevalence of IncX1 group plasmids than IncX2. The 21 kb 'genetic load' region of pOLA52 was shown to consist of a mosaic, among other things a fragmented Tn3 transposon encoding ampicillin resistance. Most notably the oqxAB and mrkABCDF cassettes were contained within two composite transposons (Tn6010...

Modeling Ebola Virus Genome Replication and Transcription with Minigenome Systems.

Science.gov (United States)

Cressey, Tessa; Brauburger, Kristina; Mühlberger, Elke

2017-01-01

In this chapter, we describe the minigenome system for Ebola virus (EBOV), which reconstitutes EBOV polymerase activity in cells and can be used to model viral genome replication and transcription. This protocol comprises all steps including cell culture, plasmid preparation, transfection, and luciferase reporter assay readout.

pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

Science.gov (United States)

Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

2018-05-01

The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

49 CFR 22.1 - Purpose.

Science.gov (United States)

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Purpose. 22.1 Section 22.1 Transportation Office of the Secretary of Transportation SHORT-TERM LENDING PROGRAM (STLP) General § 22.1 Purpose. The purpose of the DOT OSDBU STLP is to provide financial assistance in the form of short-term loans from...

The inhibition of the highly expressed miR-221 and miR-222 impairs the growth of prostate carcinoma xenografts in mice.

Directory of Open Access Journals (Sweden)

Neri Mercatelli

Full Text Available BACKGROUND: MiR-221 and miR-222 are two highly homologous microRNAs whose upregulation has been recently described in several types of human tumors, for some of which their oncogenic role was explained by the discovery of their target p27, a key cell cycle regulator. We previously showed this regulatory relationship in prostate carcinoma cell lines in vitro, underlying the role of miR-221/222 as inducers of proliferation and tumorigenicity. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a number of in vivo approaches confirming our previous data. The ectopic overexpression of miR-221 is able, per se, to confer a high growth advantage to LNCaP-derived tumors in SCID mice. Consistently, the anti-miR-221/222 antagomir treatment of established subcutaneous tumors derived from the highly aggressive PC3 cell line, naturally expressing high levels of miR-221/222, reduces tumor growth by increasing intratumoral p27 amount; this effect is long lasting, as it is detectable as long as 25 days after the treatment. Furthermore, we provide evidence in favour of a clinical relevance of the role of miR-221/222 in prostate carcinoma, by showing their general upregulation in patient-derived primary cell lines, where we find a significant inverse correlation with p27 expression. CONCLUSIONS/SIGNIFICANCE: These findings suggest that modulating miR-221/222 levels may have a therapeutic potential in prostate carcinoma.

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. Copyright © 2013 Elsevier Inc. All rights reserved.

The complete sequence and comparative analysis of a multidrug- resistance and virulence multireplicon IncFII plasmid pEC302/04 from an extraintestinal pathogenic Escherichia coli EC302/04 indicate extensive diversity of IncFII plasmids

Directory of Open Access Journals (Sweden)

Wing Sze eHo

2016-01-01

Full Text Available Extraintestinal pathogenic Escherichia coli (ExPEC that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA and FIB with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok and a plasmid partitioning system, ParAB and PsiAB, which are important for plasmid maintenance were also found.Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of

The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids.

Science.gov (United States)

Ho, Wing Sze; Yap, Kien-Pong; Yeo, Chew Chieng; Rajasekaram, Ganeswrie; Thong, Kwai Lin

2015-01-01

Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as bla TEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical

36 CFR 2.21 - Smoking.

Science.gov (United States)

2010-07-01

... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Smoking. 2.21 Section 2.21 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR RESOURCE PROTECTION, PUBLIC USE AND RECREATION § 2.21 Smoking. (a) The superintendent may designate a portion of a park area, or all or a portion of a building,...

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

Science.gov (United States)

Thomason, Lynn C; Costantino, Nina; Court, Donald L

2016-09-13

Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single

Rolling-circle plasmids from Bacillus subtilis : complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from Gram-positive bacteria

NARCIS (Netherlands)

Meijer, W.J.; Schuurs-Wisman, Bea; Terpstra, P; Thorsted, P.; Thomas, C.M.; Holsappel, S; Venema, G; Bron, S

Most small plasmids of Gram-positive bacteria use the rolling-circle mechanism of replication and several of these have been studied in considerable detail at the DNA level and for the function of their genes. Although most of the common laboratory Bacillus subtilis 168 strains do not contain

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

Plasmid DNA is released from nanosized acicular material surface by low molecular weight oligonucleotides: exogenous plasmid acquisition mechanism for penetration intermediates based on the Yoshida effect.

Science.gov (United States)

Yoshida, N; Ide, K

2008-10-01

When a colloidal solution consisting of nanosized acicular material and bacterial cells is stimulated with sliding friction at the interface between the hydrogel and interface-forming material where the frictional coefficient increases rapidly, the nanosized acicular material accompanying the bacterial cells forms a penetration intermediate. This effect is known as the Yoshida effect in honor of its discoverer. Through the Yoshida effect, a novel property in which penetration intermediates incorporate exogenous plasmid DNA has been identified. This report proposes a possible mechanism for exogenous plasmid acquisition by penetration intermediates in the Yoshida effect. Escherichia coli cells, pUC18, and chrysotile were used as recipient cells, plasmid DNA, and nanosized acicular material, respectively. Even when repeatedly washing the mixture consisting of pUC18 and chrysotile, transformation efficiency by pUC18 was stable. Accordingly, pUC18 adsorbed onto chrysotile was introduced into recipient E. coli cells. At saturation, the amount of pUC18 adsorbed onto chrysotile was 0.8-1.2 microg/mg. To investigate whether pUC18 adsorbed on chrysotile is replicated by polymerase, polymerase chain reaction (PCR) was carried out with the chrysotile. Amplification of the beta-lactamase gene coded in pUC18, which was adsorbed onto chrysotile, was strongly inhibited. This suggests that DNA adsorbed onto chrysotile is not replicated in vivo. When we searched for substances to release pUC18 adsorbed onto chrysotile, we found that a 300-bp single- or double-stranded segment of DNA releases pUC18 from chrysotile. Competitive adsorption onto chrysotile between double-stranded DNA and pUC18 was then examined through the Yoshida effect. The 310- and 603-bp double-stranded nucleotides caused 50% competitive inhibition at the same molar ratio with pUC18. Hence, the adsorbed region of pUC18 is about 300 bp in length. As the culture period for recipient cells increases, transformation

A New Catabolic Plasmid in Xanthobacter and Starkeya spp. from a 1,2-Dichloroethane-Contaminated Site

Science.gov (United States)

Munro, Jacob E.; Liew, Elissa F.; Ly, Mai-Anh

2016-01-01

ABSTRACT 1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria (Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1 and ISPme1) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1 and dhlB2) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1. Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. IMPORTANCE Earlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more

Isolation and characterization of two cryptic plasmids in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11.

Science.gov (United States)

Yamagata, A; Kato, J; Hirota, R; Kuroda, A; Ikeda, T; Takiguchi, N; Ohtake, H

1999-06-01

Two plasmids were discovered in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11, which was isolated from activated sludge. The plasmids, designated pAYS and pAYL, were relatively small, being approximately 1.9 kb long. They were cryptic plasmids, having no detectable plasmid-linked antibiotic resistance or heavy metal resistance markers. The complete nucleotide sequences of pAYS and pAYL were determined, and their physical maps were constructed. There existed two major open reading frames, ORF1 in pAYS and ORF2 in pAYL, each of which was more than 500 bp long. The predicted product of ORF2 was 28% identical to part of the replication protein of a Bacillus plasmid, pBAA1. However, no significant similarity to any known protein sequences was detected with the predicted product of ORF1. pAYS and pAYL had a highly homologous region, designated HHR, of 262 bp. The overall identity was 98% between the two nucleotide sequences. Interestingly, HHR-homologous sequences were also detected in the genomes of ENI-11 and the plasmidless strain Nitrosomonas europaea IFO14298. Deletion analysis of pAYS and pAYL indicated that HHR, together with either ORF1 or ORF2, was essential for plasmid maintenance in ENI-11. To our knowledge, pAYS and pAYL are the first plasmids found in the ammonia-oxidizing autotrophic bacteria.

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

Science.gov (United States)

Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

2014-12-01

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

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

39 CFR 221.5 - Headquarters organization.

Science.gov (United States)

2010-07-01

... 39 Postal Service 1 2010-07-01 2010-07-01 false Headquarters organization. 221.5 Section 221.5 Postal Service UNITED STATES POSTAL SERVICE ORGANIZATION AND ADMINISTRATION GENERAL ORGANIZATION § 221.5 Headquarters organization. (a) Postmaster General—(1) Appointment. The postmaster general (PMG), the chief...

49 CFR 234.221 - Lamp voltage.

Science.gov (United States)

2010-10-01

... 49 Transportation 4 2010-10-01 2010-10-01 false Lamp voltage. 234.221 Section 234.221 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION..., Inspection, and Testing Maintenance Standards § 234.221 Lamp voltage. The voltage at each lamp shall be...

Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences

Science.gov (United States)

Garcillán-Barcia, M. Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M.; de la Cruz, Fernando

2014-01-01

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ–proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages. PMID:25522143

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET, a new method for plasmid reconstruction from whole genome sequences.

Directory of Open Access Journals (Sweden)

Val F Lanza

2014-12-01

Full Text Available Bacterial whole genome sequence (WGS methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage, comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC, comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

39 CFR 221.4 - Corporate officers.

Science.gov (United States)

2010-07-01

... 39 Postal Service 1 2010-07-01 2010-07-01 false Corporate officers. 221.4 Section 221.4 Postal Service UNITED STATES POSTAL SERVICE ORGANIZATION AND ADMINISTRATION GENERAL ORGANIZATION § 221.4 Corporate officers. The Board of Governors determines the number of corporate officers and appoints the...

Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions.

Science.gov (United States)

Whiteway, Alistair; Deru, Wale; Prentice, H Grant; Anderson, Robert

2003-12-01

Recombinant adeno-associated virus type 2 (rAAV) has promise for use as a gene therapy vector. Potential problems in the production of rAAV stocks are both the limited amount of recombinant virus that is produced by traditional methods and the possibility of wild-type replication competent adeno-associated virus (wtAAV) contamination. The presence of these contaminants is largely dependent upon the helper plasmid used. Whilst wtAAV is not a pathogen, the presence of these contaminants is undesirable as they may affect experiments concerning the biology of rAAV. Additionally as protocols using rAAV with altered tropism are becoming more prevalent, it is important that no recombination be permitted that may cause the creation of a replication competent AAV with modified (targeting) capsids. Many experimental protocols require the generation of large amounts of high titre rAAV stocks. We describe the production of several AAV helper plasmids and cell lines designed to achieve this goal. These plasmids possess split AAV rep and cap genes to eliminate the production of wtAAV and they possess a selection mechanism which is operatively linked to expression from the AAV cap gene. This allows positive selection of those cells expressing the highest level of the structural capsid proteins and therefore those cells which yield the highest amount of rAAV.

Isolation and sequence analysis of pCS36-4CPA, a small plasmid from Citrobacter sp. 36-4CPA.

Science.gov (United States)

Zharikova, Natalia V; Iasakov, Timur R; Bumazhkin, Boris K; Patutina, Ekaterina O; Zhurenko, Evgeniia I; Korobov, Vladislav V; Sagitova, Alina I; Kuznetsov, Boris B; Markusheva, Tatiana V

2018-05-01

A small plasmid designated pCS36-4CPA with a size of 5217 base pairs and G-C content of 50.74% was isolated from Citrobacter sp. 36-4CPA. The origin of replication ( ori ) of the plasmid was identified as a region of about 800 bp in length with an identity of 67.1% to the ColE1 plasmid at the nucleotide level. The replication region contained typical elements of ColE1-like plasmids: RNA I and RNA II with their corresponding -10 and -35 boxes, a single-strand initiation site ( ssi ), and a lagging-strand termination site ( terH ). As seen in other ColE1-like plasmids, pCS36-4CPA carried mobilisation machinery that include mobABCD genes but it did not possess the rom gene. Analysis of the multimer resolution site ( mrs ) was performed and XerC and XerD binding sites were identified. Also, the 70-nt transcript Rcd of pCS36-4CPA was predicted and similarity of the transcript's secondary structure with those of the ColE1-family was shown. The cargo module of pCS36-4CPA contained three open reading frames (ORFs). Two of them (ORF5 and ORF6) showed no significant homology to any known gene sequences but contained putative THAP DNA-binding (DBD) and type II restriction endonuclease Eco O109I domains. The seventh open reading frame (ORF7) encodes YhdJ-like DNA modification methylase. The region highly homologous to pCS36-4CPA was found in the Salmonella phage SE2 genome.

14 CFR 221.4 - English language.

Science.gov (United States)

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false English language. 221.4 Section 221.4... REGULATIONS TARIFFS General § 221.4 English language. All tariffs and other documents and material filed with the Department pursuant to this part shall be in the English language. ...

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.

Fermentation of starch by Klebsiella oxytoca P2, containing plasmids with {alpha}-amylase and pullulanase genes

Energy Technology Data Exchange (ETDEWEB)

Santos, V.L. dos; Araujo, E.F.; Barros, E.G. de; Guimaraes, W.V.

1999-12-20

Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12--24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower {alpha}-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.

24 CFR 221.1 - Savings clause.

Science.gov (United States)

2010-04-01

... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Savings clause. 221.1 Section 221.1... MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES LOW COST AND MODERATE INCOME MORTGAGE INSURANCE-SAVINGS CLAUSE Eligibility Requirements-Low Cost Homes-Savings Clause § 221.1...

24 CFR 221.775 - Option period.

Science.gov (United States)

2010-04-01

... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Option period. 221.775 Section 221.775 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued... § 221.775 Option period. The mortgagee may exercise its option to assign within one year following the...

Identification of pOENI-1 and Related Plasmids in Oenococcus oeni Strains Performing the Malolactic Fermentation in Wine

Science.gov (United States)

Favier, Marion; Bilhère, Eric; Lonvaud-Funel, Aline; Moine, Virginie; Lucas, Patrick M.

2012-01-01

Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly

22 CFR 221.43 - Governing law.

Science.gov (United States)

2010-04-01

... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Governing law. 221.43 Section 221.43 Foreign... Administration § 221.43 Governing law. This Guarantee shall be governed by and construed in accordance with the laws of the United States of America governing contracts and commercial transactions of the United...

46 CFR 108.221 - Storm rails.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Storm rails. 108.221 Section 108.221 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Construction and Arrangement Rails § 108.221 Storm rails. Each unit must have a storm rail in the following...

49 CFR 221.7 - Civil penalty.

Science.gov (United States)

2010-10-01

... 49 Transportation 4 2010-10-01 2010-10-01 false Civil penalty. 221.7 Section 221.7 Transportation... TRANSPORTATION REAR END MARKING DEVICE-PASSENGER, COMMUTER AND FREIGHT TRAINS General § 221.7 Civil penalty. Any... requirement is subject to a civil penalty of at least $650 and not more than $25,000 per violation, except...

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

2017-05-01

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. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Influence of Expression Plasmid of Connective Tissue Growth Factor and Tissue Inhibitor of Metalloproteinase-1 shRNA on Hepatic Precancerous Fibrosis in Rats.

Science.gov (United States)

Zhang, Qun; Shu, Fu-Li; Jiang, Yu-Feng; Huang, Xin-En

2015-01-01

In this study, influence caused by expression plasmids of connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) short hairpin RNA (shRNA) on mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII in hepatic tissue with hepatic fibrosis, a precancerous condition, in rats is analyzed. To screen and construct shRNA expression plasimid which effectively interferes RNA targets of CTGF and TIMP-1 in rats. 50 cleaning Wistar male rats are allocated randomly at 5 different groups after precancerous fibrosis models and then injection of shRNA expression plasimids. Plasmid psiRNA-GFP-Com (CTGF and TIMP-1 included), psiRNA-GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA- DUO-GFPzeo of blank plasmid are injected at group A, B, C and D, respectively, and as model control group that none plasimid is injected at group E. In 2 weeks after last injection, to hepatic tissue at different groups, protein expression of CTGF, TIMP-1, procol-α1and PC III is tested by immunohistochemical method and,mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII is measured by real-time PCR. One-way ANOVA is used to comparison between-groups. Compared with model group, there is no obvious difference of mRNA expression among CTGF,TIMP-1,procol-α1,PC III and of protein expression among CTGF, TIMP-1, procol-α1, PC III in hepatic tissue at group injected with blank plasmid. Expression quantity of mRNA of CTGF, TIMP-1, procol-α1 and PCIII at group A, B and C decreases, protein expression of CTGF, TIMP-1, procol-α1, PC III in hepatic tissue is lower, where the inhibition of combination RNA interference group (group A) on procol-α1 mRNA transcription and procol-α1 protein expression is superior to that of single interference group (group B and C) (P<0.01 or P<0.05). RNA interference on CTGF and/or TIMP-1 is obviously a inhibiting factor for mRNA and protein expression of CTGF, TIMP-1, procol-α1 and PCIII. Combination RNA interference on genes of CTGF and TIMP-1 is superior

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 ...

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.

Origin and Evolution of Rickettsial Plasmids.

Directory of Open Access Journals (Sweden)

Khalid El Karkouri

Full Text Available 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

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

International Nuclear Information System (INIS)

Polonskaya, Zhanna; Benham, Craig J.; Hearing, Janet

2004-01-01

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component of oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication

49 CFR 221.3 - Application.

Science.gov (United States)

2010-10-01

... transportation; (3) Rapid transit operations in an urban area that are not connected with the general railroad... 49 Transportation 4 2010-10-01 2010-10-01 false Application. 221.3 Section 221.3 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF...

ORPLOT.PC: a graphic utility for ORMGEN.PC and ORVIRT.PC

International Nuclear Information System (INIS)

Inversini, C.; Bryson, J.W.

1986-06-01

ORPLOT.PC is an interactive graphic utility for ORMGEN.PC and ORVIRT.PC. It executes on an IBM PC/XT or PC/AT equipped with hard disk, graphic card, and 512K minimum memory. The program is capable of: (1) displaying finite-element meshes generated by ORMGEN.PC complete with node numbers, element numbers, and boundary conditions; and (2) generating deformed mesh plots, contour plots, line (X-Y) plots, and developed surface plots of ORVIRT.PC output. A zooming feature allows detailed inspection of any subregion. Because simplicity and ease of use were important objectives during program development, all commands are entered interactively using free format. The option of automatic or user-defined scaling for most plots is another convenience. Plot files may be created and written to hard disk for subsequent hardcopy to printer or plotter. 2 refs., 7 figs

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

OpenAIRE

Serror, Pascale; Sasaki, Takashi; Ehrlich, S. Dusko; Maguin, Emmanuelle

2002-01-01

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 104 transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a goo...

20 CFR 221.1 - Introduction.

Science.gov (United States)

2010-04-01

... 270 of this chapter). The Board is responsible for making this decision. ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Introduction. 221.1 Section 221.1 Employees... Security Administration or the Railroad Retirement Board will pay benefits to a railroad employee, and his...

The construction and identification of hypoxia-regulated recombinant plasmid with reporter gene hNIS

International Nuclear Information System (INIS)

Hu Qunchao; Wu Jinchang; Zhou Jundong; Gu Ke

2011-01-01

Objective: To construct pShuttle-5 × HRE-CMV-NIS recombinant plasmid regulated by hypoxia-responsive element, which can possibly by used to detect the expression of hypoxia induced factor-α (HIF-1α) gene under hypoxia condition. Methods: Artificially synthesize the nucleotide sequences of five copies of hypoxia response elements (HREs) were cloned into pGL3-promoter vector to construct pGL3-promoter-5 × HRE vector. Human sodium/iodide symporter (hNIS) gene cDNA was amplified from human genome by RT-PCR, and subcloned into pGL3-promoter-5 × HRE vector then was sequenced. After treated with CoCl 2 as hypoxia mimic, HEK293 cells were transfected with recombinant plasmid with hNIS gene, while cells treated with DMSO as the control. Meanwhile, pcDNA3.1-HIF-1α and recombinant hNIS gene vectors were transfected into HEK293 cells at the ratio of 3 to 1, while co-transfection with pcDNA3.1 and pShuttle-NIS vectors cells were taken as the control. NIS mRNA expression was analyzed by qRT-PCR while function of NIS protein was tested by 99m TcO 4 - -uptake. Results: The sequence data of hNIS gene in recombinant plasmid were in accordance with those reported in the literatures. Compared with control groups, HEK293 cells co-transfected with both pShuttle-5 × HRE-CMV-NIS and HIF-1α gene vectors and CoCl 2 -treated after pShuttle-NIS transfecting presented higher mRNA expressions of NIS and 99m TcO 4 - uptake (P<0.01). Conclusion: HIF-1α can be bound to and activate pShuttle-5 × HRE-CMV-NIS in cells to accumulate radioactive nuclide 99m TcO 4 - and this technique is potential for detection of expression and activity of HIF-1α, the indicator of cell hypoxia. (authors)

39 CFR 221.2 - Board of Governors.

Science.gov (United States)

2010-07-01

... 39 Postal Service 1 2010-07-01 2010-07-01 false Board of Governors. 221.2 Section 221.2 Postal Service UNITED STATES POSTAL SERVICE ORGANIZATION AND ADMINISTRATION GENERAL ORGANIZATION § 221.2 Board of Governors. (a) Composition. The Board of Governors consists of 11 members. Nine governors are appointed by...

Replication of chromosomal and episomal DNA in X-ray-damaged human cells: A cis- or trans-acting mechanism

International Nuclear Information System (INIS)

Cleaver, J.E.; Rose, R.; Mitchell, D.L.

1990-01-01

Episomal plasmids and viruses in mammalian cells present small targets for X-ray-induced DNA damage. At doses up to 100 Gy, DNA strand breaks or endonuclease III-sensitive sites were not discernible in 10.3-kb Epstein-Barr virus-based plasmid DNA or in 4.9-kb defective simian virus 40 DNA. DNA replication in these small molecules, however, was inhibited strongly by X-ray doses of greater than or equal to 20 Gy, decreasing to only 20 to 40% of control values. Inhibition was relieved slightly by growth in caffeine but was increased by growth in 3-aminobenzamide. Inhibition of DNA replication in episomal DNA molecules that are too small to sustain significant damage directly to their DNA may be due to either (a) a trans-acting diffusible factor that transfers the consequences of DNA breakage to episomes and to other replicating molecules, (b) a cis-acting mechanism in which episomes are structurally linked to genomic chromatin, and replication of both episomal and chromosomal replicons is under common control, or (c) radiation damage on other cellular structures unrelated to DNA. The resolution of these cellular mechanisms may shed light on the X-ray-resistant replication in ataxia-telangiectasia and may suggest strategies for molecular characterization of potential trans- or cis-acting factors

STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE SINGLE-STRAND ORIGIN OF REPLICATION FROM THE LACTOCOCCAL PLASMID PWVO1

NARCIS (Netherlands)

SEEGERS, JFML; ZHAO, AC; MEIJER, WJJ; KHAN, SA; VENEMA, G; BRON, S

1995-01-01

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on

Yeast transformation mediated by Agrobacterium strains harboring an Ri plasmid: comparative study between GALLS of an Ri plasmid and virE of a Ti plasmid.

Science.gov (United States)

Kiyokawa, Kazuya; Yamamoto, Shinji; Sato, Yukari; Momota, Naoto; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

2012-07-01

Agrobacterium strains containing a Ti plasmid can transfer T-DNA not only to plants but also to fungi, including the yeast Saccharomyces cerevisiae. However, no Agrobacterium strain harboring an Ri plasmid has been evaluated in fungal transformation. Some Ri plasmids have GALLS , instead of virE1 and virE2. GALLS protein can functionally substitute in plant transformation for a structurally different protein VirE2. In this study, we compared the yeast transformation ability among Agrobacterium donors: a strain containing a Ti plasmid, strains harboring either an agropine-type or a mikimopine-type Ri plasmid, and a strain having a modified Ri plasmid supplemented with a Ti plasmid type virE operon. Agrobacterium strains possessing GALLS transformed yeast cells far less efficiently than the strain containing virE operon. Production of GALLS in recipient yeast cells improved the yeast transformation mediated by an Agrobacterium strain lacking neither GALLS nor virE operon. A reporter assay to detect mobilization of the proteins fused with Cre recombinase revealed that VirE2 protein is much more abundant in yeast cells than GALLS. Based on these results, we concluded that the low yeast transformability mediated by Agrobacterium strains having the Ri plasmid is because of low amount of mobilized GALLS in yeast cells. © 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

Science.gov (United States)

Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc

2002-01-01

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.

Murine leukemia virus (MLV replication monitored with fluorescent proteins

Directory of Open Access Journals (Sweden)

Bittner Alexandra

2004-12-01

Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

Adding to Yersinia enterocolitica Gene Pool Diversity: Two Cryptic Plasmids from a Biotype 1A Isolate

Directory of Open Access Journals (Sweden)

Daniela Lepka

2009-01-01

Full Text Available We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs carried by a Yersinia enterocolitica biotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs encoding the first CcdA/CcdB-like antitoxin/toxin system described for a Yersinia plasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described in Salmonella (CcdA/B, Klebsiella (RepA, and Plesiomonas (MobA/C indicating genomic fluidity among members of the Enterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9% was similar to that of pYe4449-1 (53.7% and differed from that of the Y. enterocolitica genome (47.3%. Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at 4°C but not at or above 27°C suggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing the DxxGN(xnDxxGN motif. Our findings illustrate the exceptional gene pool diversity within the species Y. enterocolitica driven by horizontal gene transfer events.

CARTOGRAPHIE DU PLASMIDE pSU100, PLASMIDE CRYPTIQUE DE LACTOBACILLUS CASEI

Directory of Open Access Journals (Sweden)

F BENSALAH

2003-06-01

Ce plasmide appelé pSU100 a été cloné dans le vecteur de transformation pUC18 au site EcoRI chez E. coli JM103. Les profils électrophorétiques de restriction obtenus par des digestions simples, doubles et triples sous l’action de 33 endonucléases, ont contribué à l’élaboration d’une carte de restriction de ce plasmide. Cinq sites uniques ont été identifiés, ainsi que d’autres sites doubles et multiples. Une étude préliminaire du rôle physiologique de ce plasmide a permis de déceler une résistance à la kanamycine.

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.

Alpha decay 225 Ac → 221Fr

International Nuclear Information System (INIS)

Gromov, K. Ya.; Gorozhankin, V.M.; Malov, L.A.; Fominykh, V.I.; Tsupko-Sitnikov, V.V.; Chumin, V.G.; Jakushev, E.A.; Kudrya, S.A.; Sergienko, V.A.; Malikov, Sh.R.

2004-01-01

Full text: Considerable attention has been given to nuclei with A = 220 - 230 recently. In this region there occurs transition from the spherical to the deformed nuclear shape, which gives rise to some specific features in the nuclear structure. In particular, negative parity levels with low excitation energies have been found in even-even nuclei from this region [1, 2]. One of the nuclei allowing experimental investigation of the above properties is 221 Fr. The nuclide 221 Fr is from the region of isotopes which does not include stable nuclei and thus it cannot be studied in several-nucleon transfer reactions. In addition, the neutron excess in this nucleus makes it impossible to study the nucleus in reactions with heavy ions. Experimental information on the 221 Fr level structure can only be gained from investigation of the 225 Ac (T 1/2 = 10 days) alpha decay or the 221 Rn (T 1/2 = 25 min) beta decay. In the latter case the possibilities of the investigation are restricted by difficulties in making of 221 Rn sources. Therefore, most information on the structure and properties of 221 Fr is derived from investigation of the 225 Ac α -decay [3]. In-depth investigation of ( α - γ )- coincidences at the 225 Ac decay is carried out. Twenty-one new weak γ - rays are found; 18 γ-rays earlier ascribed to the 225 Ac decay are not confirmed. The quantitative analysis of the ( α - γ )- coincidences makes it possible to find the intensity of 221 Fr levels by the decay and multipolarities of five weak γ -transitions. The conversion electron spectrum is investigated in the range of 5 † 24 keV with a high (some 20 eV) energy resolution. A new M1 type 10.6-keV γ-transition is found. The proposed 225 Ac decay scheme includes 31 excited 221 Fr states. Parities are established for 16 of them. Possible spin values are proposed for 221 Fr levels. Properties of excited 221 Fr states are satisfactorily described by the quasiparticle-phonon nuclear model without the

Large-scale preparation of plasmid DNA.

Science.gov (United States)

Heilig, J S; Elbing, K L; Brent, R

2001-05-01

Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

Microarray-based analysis of IncA/C plasmid-associated genes from multidrug-resistant Salmonella enterica.

Science.gov (United States)

Lindsey, Rebecca L; Frye, Jonathan G; Fedorka-Cray, Paula J; Meinersmann, Richard J

2011-10-01

In the family Enterobacteriaceae, plasmids have been classified according to 27 incompatibility (Inc) or replicon types that are based on the inability of different plasmids with the same replication mechanism to coexist in the same cell. Certain replicon types such as IncA/C are associated with multidrug resistance (MDR). We developed a microarray that contains 286 unique 70-mer oligonucleotide probes based on sequences from five IncA/C plasmids: pYR1 (Yersinia ruckeri), pPIP1202 (Yersinia pestis), pP99-018 (Photobacterium damselae), pSN254 (Salmonella enterica serovar Newport), and pP91278 (Photobacterium damselae). DNA from 59 Salmonella enterica isolates was hybridized to the microarray and analyzed for the presence or absence of genes. These isolates represented 17 serovars from 14 different animal hosts and from different geographical regions in the United States. Qualitative cluster analysis was performed using CLUSTER 3.0 to group microarray hybridization results. We found that IncA/C plasmids occurred in two lineages distinguished by a major insertion-deletion (indel) region that contains genes encoding mostly hypothetical proteins. The most variable genes were represented by transposon-associated genes as well as four antimicrobial resistance genes (aphA, merP, merA, and aadA). Sixteen mercury resistance genes were identified and highly conserved, suggesting that mercury ion-related exposure is a stronger pressure than anticipated. We used these data to construct a core IncA/C genome and an accessory genome. The results of our studies suggest that the transfer of antimicrobial resistance determinants by transfer of IncA/C plasmids is somewhat less common than exchange within the plasmids orchestrated by transposable elements, such as transposons, integrating and conjugative elements (ICEs), and insertion sequence common regions (ISCRs), and thus pose less opportunity for exchange of antimicrobial resistance.

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...

Evaluation of the PSMA promoter for use in a conditionally replicative adenovirus (CRAd) and radiotherapy

International Nuclear Information System (INIS)

Ove, R.; Swift, A.; Kransnykh, V.N.; Nettelbeck, D.M.; Yamamato, M.; Curiel, D.T.

2003-01-01

CRAds are currently being evaluated for possible use with concurrent prostate radiotherapy/brachytherapy. Viruses with tissue specific promoters governing the viral E1A region exist for Cox-2 [Yamamato] and Flt-1 Tyr[Nettelbeck], and a PSMA (prostate specific membrane antigen) promoter and promoter/enhancer CRAd is being developed. PSMA is promising in that it is very specific to prostate cancer. We evaluated replication deficient Cox-2 and PSMA luciferase reporter viruses (Ad-Cox2-luc and Ad-PSMA-luc/ RGD) with and without 3Gy radiation. The Ad-PSMA-luc/RGD genome contains an RGD fiber knob modification, to allow infection via integrin interaction (bypassing CAR). Cell lines used were the prostate cell lines PC3 (PSMA-) and LNCaP (PSMA+), the breast line BT474 (Cox2-), and immortalized human hepatocytes (THLE-3). Cells were radiated 1 hour before viral infection, and assayed 48-hours after infection. Luciferase activity was assayed per total cellular protein of the surviving cells. Ad-PSMA-luc was negative in BT474, THLE3, and PC3, but showed increased expression in LNCaP. Radiation moderately increased expression. Ad-Cox2-luc was positive in all four lines. Radiation led to slightly lower promoter activity in BT474 and PC3, 2-fold lower expression in LNCaP, and a slight increase in THLE3. A control reporter virus (Ad-CMV-luc) was strongly positive for all lines except BT474, which was negative. Radiation had no affect on CMV promoter activity. Comparison PSMA promoter and PSMA promoter/enhancer plasmids showed a 60-fold increase with the addition of the enhancer (50 times the SV40 promoter/enhancer). The enhancer led to only 2-fold increase in Du145 (PSMA-). The PSMA reporter virus (Ad-PSMA-luc) showed selective activity in PSMA positive cells. Moderate enhancement is seen with low dose radiation, but not with Ad-Cox2-luc. The PSMA promoter and promoter/enhancer are promising in the CRAd context. Timing and dose of irradiation needs to be explored further

46 CFR 221.93 - Collection of civil penalties.

Science.gov (United States)

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Collection of civil penalties. 221.93 Section 221.93... RELATED ACTIVITIES REGULATED TRANSACTIONS INVOLVING DOCUMENTED VESSELS AND OTHER MARITIME INTERESTS Civil Penalties § 221.93 Collection of civil penalties. Within 30 days after receipt of the Hearing Officer's...

42 CFR 93.221 - PHS support.

Science.gov (United States)

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false PHS support. 93.221 Section 93.221 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES HEALTH ASSESSMENTS AND HEALTH EFFECTS STUDIES OF HAZARDOUS SUBSTANCES RELEASES AND FACILITIES PUBLIC HEALTH SERVICE POLICIES ON RESEARCH...

29 CFR 1952.221 - Developmental schedule.

Science.gov (United States)

2010-07-01

... 29 Labor 9 2010-07-01 2010-07-01 false Developmental schedule. 1952.221 Section 1952.221 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR... Management data system operational July 1, 1973. Automated Management data system operational January 1, 1974...

Alpha decay 225Ac→221Fr

International Nuclear Information System (INIS)

Kudrya, S.A.; Sergienko, V.A.; Gorozhankin, V.M.; Gromov, K.Ya.; Malikov, Sh.R.; Malov, L.A.; Fominykh, V.I.; Tsupko-Sitnikov, V.V.; Chumin, V.G.; Yakushev, E.A.

2002-01-01

In-depth investigation of (α-γ)-coincidences at the 225 Ac decay is carried out. Twenty-one new weak γ-rays are found; 18 γ-rays earlier ascribed to the 225 Ac decay are not confirmed. The quantitative analysis of the (α-γ)-coincidences makes it possible to find the intensity of population of 221 Fr levels by the α decay and multipolarities of five weak γ-transitions. The conversion electron spectrum is investigated in the range of 5-24 keV with a high (some 20 eV) energy resolution. A new M1 type 10.6-keV γ-transition is found. The proposed 225 Ac decay scheme includes 31 excited 221 Fr states. Parities are established for 16 of them. Possible spin values are proposed for 221 Fr levels. Properties of excited 221 Fr states are satisfactorily described by the quasiparticle-phonon nuclear model without the assumption of static octupole deformation

Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

DEFF Research Database (Denmark)

Johnson, Timothy J.; Bielak, Eliza Maria; Fortini, Daniela

2012-01-01

and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid...

Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places.

Science.gov (United States)

van Brabant, A J; Hunt, S Y; Fangman, W L; Brewer, B J

1998-06-01

DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

27 CFR 25.221 - Voluntary destruction of beer.

Science.gov (United States)

2010-04-01

... beer. 25.221 Section 25.221 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS BEER Voluntary Destruction § 25.221 Voluntary destruction of beer. (a) On brewery premises. (1) A brewer may destroy, at the brewery, beer on which the tax has not...

24 CFR 221.770 - Assignment option.

Science.gov (United States)

2010-04-01

... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Assignment option. 221.770 Section... § 221.770 Assignment option. A mortgagee holding a conditional or firm commitment issued on or before... mortgagee's approved underwriter on or before November 30, 1983) has the option to assign, transfer and...

10 CFR 221.11 - Natural gas and ethane.

Science.gov (United States)

2010-01-01

... 10 Energy 3 2010-01-01 2010-01-01 false Natural gas and ethane. 221.11 Section 221.11 Energy DEPARTMENT OF ENERGY OIL PRIORITY SUPPLY OF CRUDE OIL AND PETROLEUM PRODUCTS TO THE DEPARTMENT OF DEFENSE UNDER THE DEFENSE PRODUCTION ACT Exclusions § 221.11 Natural gas and ethane. The supply of natural gas...

Impact of co-carriage of IncA/C plasmids with additional plasmids on the transfer of antimicrobial resistance in Salmonella enterica isolates.

Science.gov (United States)

Han, Jing; Pendleton, Sean J; Deck, Joanna; Singh, Ruby; Gilbert, Jeffrey; Johnson, Timothy J; Sanad, Yasser M; Nayak, Rajesh; Foley, Steven L

2018-04-20

Antimicrobial resistance in Salmonella enterica is often plasmid encoded. A key resistance plasmid group is the incompatibility group (Inc) A/C plasmids that often carry multiple resistance determinants. Previous studies showed that IncA/C plasmids were often co-located with other plasmids. The current study was undertaken to evaluate the impact of plasmid co-carriage on antimicrobial resistance and plasmid transfer. A total of 1267 Salmonella isolates, representing multiple serotypes and sources were previously subjected to susceptibility testing and 251 isolates with resistance to at least 5 antimicrobial agents were identified for further study. Each isolate was subjected to PCR-based replicon typing, and those with IncA/C plasmids were selected for plasmid isolation, PCR-based mapping of IncA/C plasmid backbone genes, and conjugation assays to evaluate resistance plasmid transferability. Of the 87 identified IncA/C positive isolates, approximately 75% carried a plasmid with another identified replicon type, with the most common being I1 (39%), FIA, FIIA, FIB and HI2 (each 15%). PCR-based mapping indicated significant diversity in IncA/C backbone content, especially in regions encoding transfer-associated and hypothetical proteins. Conjugation experiments showed that nearly 68% of the isolates transferred resistance plasmids, with 90% containing additional identified plasmids or larger (>50 kb) non-typeable plasmids. The majority of IncA/C-positive strains were able to conjugally transfer antimicrobial resistance to the recipient, encoded by IncA/C and/or co-carried plasmids. These findings highlight the importance of co-located plasmids for resistance dissemination either by directly transferring resistance genes or by potentially providing the needed conjugation machinery for IncA/C plasmid transfer. Copyright © 2018. Published by Elsevier B.V.

Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

Science.gov (United States)

Ohbayashi, Ryudo; Watanabe, Satoru; Ehira, Shigeki; Kanesaki, Yu; Chibazakura, Taku; Yoshikawa, Hirofumi

2016-05-01

Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.

Characterization of new plasmids from methylotrophic bacteria.

Science.gov (United States)

Brenner, V; Holubová, I; Benada, O; Hubácek, J

1991-07-01

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.

29 CFR 778.221 - “Call-back” pay.

Science.gov (United States)

2010-07-01

... 29 Labor 3 2010-07-01 2010-07-01 false âCall-backâ pay. 778.221 Section 778.221 Labor Regulations...Regular Rateâ Payments Not for Hours Worked § 778.221 “Call-back” pay. (a) General. In the interest of... payments consist of a specified number of hours' pay at the applicable straight time or overtime rates...

Explanatory chapter: how plasmid preparation kits work.

Science.gov (United States)

Koontz, Laura

2013-01-01

To isolate plasmid DNA from bacteria using a commercial plasmid miniprep kit (if interested, compare this protocol with Isolation of plasmid DNA from bacteria). Copyright © 2013 Elsevier Inc. All rights reserved.

PC communication

International Nuclear Information System (INIS)

Oh, Jae Cheol

1992-03-01

This text book is comprised of five charters, which is about PC communication for beginners who need to learn manners and how to use Ketel and PC serve. So it introduces first, conception of PC and precautions on using PC communication, second, preparation for PC communication with Modem, its program, install, kinds of protocol and how to use protocol, third directions of emulator of PC communication and super session, fourth, instruction of Ketel with join and access, basic command of Ketel, list of Ketel's menu, Ketel editor, service guide, directions of News service, Stock and bond service business and economic figures, exchange rate and interest rate, tax culture and leisure, Ketel BBS service and posting. The last part has a instruction of PC-serve about join, basic command of PC-serve, service guide and practical guideline.

PC communication

Energy Technology Data Exchange (ETDEWEB)

Oh, Jae Cheol

1992-03-15

This text book is comprised of five charters, which is about PC communication for beginners who need to learn manners and how to use Ketel and PC serve. So it introduces first, conception of PC and precautions on using PC communication, second, preparation for PC communication with Modem, its program, install, kinds of protocol and how to use protocol, third directions of emulator of PC communication and super session, fourth, instruction of Ketel with join and access, basic command of Ketel, list of Ketel's menu, Ketel editor, service guide, directions of News service, Stock and bond service business and economic figures, exchange rate and interest rate, tax culture and leisure, Ketel BBS service and posting. The last part has a instruction of PC-serve about join, basic command of PC-serve, service guide and practical guideline.

A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication

International Nuclear Information System (INIS)

Ohsaki, Eriko; Ueda, Keiji

2017-01-01

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. - Highlights: •KSHV replication in latency depends on LANA localization to the nuclear matrix. •LANA DBD was fused with NuMA, a nuclear matrix protein, at the N- and C-terminus. •NuMA-DBD was in the nuclear matrix and supported the ori-P dependent replication. •LANA in the nuclear matrix should be important for the KSHV replication in latency.

A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication

Energy Technology Data Exchange (ETDEWEB)

Ohsaki, Eriko; Ueda, Keiji, E-mail: kueda@virus.med.osaka-u.ac.jp

2017-01-15

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. - Highlights: •KSHV replication in latency depends on LANA localization to the nuclear matrix. •LANA DBD was fused with NuMA, a nuclear matrix protein, at the N- and C-terminus. •NuMA-DBD was in the nuclear matrix and supported the ori-P dependent replication. •LANA in the nuclear matrix should be important for the KSHV replication in latency.

Factors influencing microinjection molding replication quality

Science.gov (United States)

Vera, Julie; Brulez, Anne-Catherine; Contraires, Elise; Larochette, Mathieu; Trannoy-Orban, Nathalie; Pignon, Maxime; Mauclair, Cyril; Valette, Stéphane; Benayoun, Stéphane

2018-01-01

In recent years, there has been increased interest in producing and providing high-precision plastic parts that can be manufactured by microinjection molding: gears, pumps, optical grating elements, and so on. For all of these applications, the replication quality is essential. This study has two goals: (1) fabrication of high-precision parts using the conventional injection molding machine; (2) identification of robust parameters that ensure production quality. Thus, different technological solutions have been used: cavity vacuuming and the use of a mold coated with DLC or CrN deposits. AFM and SEM analyses were carried out to characterize the replication profile. The replication quality was studied in terms of the process parameters, coated and uncoated molds and crystallinity of the polymer. Specific studies were processed to quantify the replicability of injection molded parts (ABS, PC and PP). Analysis of the Taguchi experimental designs permits prioritization of the impact of each parameter on the replication quality. A discussion taking into account these new parameters and the thermal and spreading properties on the coatings is proposed. It appeared that, in general, increasing the mold temperature improves the molten polymer fill in submicron features except for the steel insert (for which the presence of a vacuum is the most important factor). Moreover, the DLC coating was the best coating to increase the quality of the replication. This result could be explained by the lower thermal diffusivity of this coating. We noted that the viscosity of the polymers is not a primordial factor of the replication quality.

Plasmid-mediated UV-protection in Streptococcus lactis

Energy Technology Data Exchange (ETDEWEB)

Chopin, M.C.; Rouault, A. (Institut National de la Recherche Agronomique, Rennes (France). Lab. de Recherches de Technologie Laitiere); Moillo-Batt, A. (Institut National de la Sante et de la Recherche Medicale (INSERM), Hopital de Pontchaillon, 35 - Rennes (France))

1985-02-01

Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by co-transfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci.

Plasmid-mediated UV-protection in Streptococcus lactis

International Nuclear Information System (INIS)

Chopin, M.-C.; Rouault, A.

1985-01-01

Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by cotransfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci. (orig.)

49 CFR 1542.221 - Records of law enforcement response.

Science.gov (United States)

2010-10-01

... 49 Transportation 9 2010-10-01 2010-10-01 false Records of law enforcement response. 1542.221 Section 1542.221 Transportation Other Regulations Relating to Transportation (Continued) TRANSPORTATION SECURITY ADMINISTRATION, DEPARTMENT OF HOMELAND SECURITY CIVIL AVIATION SECURITY AIRPORT SECURITY Operations § 1542.221 Records of law enforcement...

Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe

Science.gov (United States)

Cotobal, Cristina; Segurado, Mónica; Antequera, Francisco

2010-01-01

DNA replication origins (ORI) in Schizosaccharomyces pombe colocalize with adenine and thymine (A+T)-rich regions, and earlier analyses have established a size from 0.5 to over 3 kb for a DNA fragment to drive replication in plasmid assays. We have asked what are the requirements for ORI function in the chromosomal context. By designing artificial ORIs, we have found that A+T-rich fragments as short as 100 bp without homology to S. pombe DNA are able to initiate replication in the genome. On the other hand, functional dissection of endogenous ORIs has revealed that some of them span a few kilobases and include several modules that may be as short as 25–30 contiguous A+Ts capable of initiating replication from ectopic chromosome positions. The search for elements with these characteristics across the genome has uncovered an earlier unnoticed class of low-efficiency ORIs that fire late during S phase. These results indicate that ORI specification and dynamics varies widely in S. pombe, ranging from very short elements to large regions reminiscent of replication initiation zones in mammals. PMID:20094030

Behavior of IncQ Plasmids in Agrobacterium tumefaciens

NARCIS (Netherlands)

Hille, Jacques; Schilperoort, Rob

1981-01-01

Inc-Q plasmids were introduced into Agrobacterium tumefuciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results

39 CFR 221.1 - The United States Postal Service.

Science.gov (United States)

2010-07-01

... 39 Postal Service 1 2010-07-01 2010-07-01 false The United States Postal Service. 221.1 Section 221.1 Postal Service UNITED STATES POSTAL SERVICE ORGANIZATION AND ADMINISTRATION GENERAL ORGANIZATION § 221.1 The United States Postal Service. The United States Postal Service was established as an...

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 suc...

40 CFR 221.1 - Applications for permits.

Science.gov (United States)

2010-07-01

... of the proposed dumping on the marine environment, navigation, living and non-living marine resource... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Applications for permits. 221.1 Section 221.1 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN DUMPING...

14 CFR 221.5 - Unauthorized air transportation.

Science.gov (United States)

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Unauthorized air transportation. 221.5... PROCEEDINGS) ECONOMIC REGULATIONS TARIFFS General § 221.5 Unauthorized air transportation. Tariff publications... Department's granting authority to perform the foreign air transportation covered by such tariff publications...

High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

Directory of Open Access Journals (Sweden)

Qureshi Nasib

2006-05-01

Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

Plasmid stability in dried cells of the desert cyanobacterium Chroococcidiopsis and its potential for GFP imaging of survivors on Earth and in space.

Science.gov (United States)

Billi, Daniela

2012-06-01

Two GFP-based plasmids, namely pTTQ18-GFP-pDU1(mini) and pDUCA7-GFP, of about 7 kbp and 15 kbp respectively, able to replicate in Chroococcidiopsis sp. CCMEE 029 and CCMEE 123, were developed. Both plasmids were maintained in Chroococcidiopsis cells after 18 months of dry storage as demonstrated by colony PCR, plasmid restriction analysis, GFP imaging and colony-forming ability under selection of dried transformants; thus suggesting that strategies employed by this cyanobacterium to stabilize dried chromosomal DNA, must have protected plasmid DNA. The suitability of pDU1(mini)-plasmid for GFP tagging in Chroococcidiopsis was investigated by using the RecA homolog of Synechocystis sp. PCC 6803. After 2 months of dry storage, the presence of dried cells with a GFP-RecA(Syn) distribution resembling that of hydrated cells, supported its capability of preventing desiccation-induced genome damage, whereas the rewetted cells with filamentous GFP-RecA(Syn) structures revealed sub-lethal DNA damage. The long-term stability of plasmid DNA in dried Chroococcidiopsis has implication for space research, for example when investigating the recovery of dried cells after Martian and space simulations or when developing life support systems based on phototrophs with genetically enhanced stress tolerance and stored in the dry state for prolonged periods.

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

Science.gov (United States)

Serror, Pascale; Sasaki, Takashi; Ehrlich, S. Dusko; Maguin, Emmanuelle

2002-01-01

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 104 transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii. PMID:11772607

Plasmid-mediated mineralization of 4-chlorobiphenyl

International Nuclear Information System (INIS)

Shields, M.S.; Hooper, S.W.; Sayler, G.S.

1985-01-01

Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 x 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14 C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB

Construction of Biologically Functional Bacterial Plasmids In Vitro

Science.gov (United States)

Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Helling, Robert B.

1973-01-01

The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins. Images PMID:4594039

The transcription factors CREB and c-Fos play key roles in NCAM-mediated neuritogenesis in PC12-E2 cells

DEFF Research Database (Denmark)

Jessen, U; Novitskaya, V; Pedersen, N

2001-01-01

The neural cell adhesion molecule (NCAM) stimulates axonal outgrowth by activation of the Ras-mitogen activated protein kinase (MAPK) pathway and by generation of arachidonic acid. We investigated whether the transcription factors, cyclic-AMP response-element binding protein (CREB) and c-Fos play...... roles in this process by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in co-culture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding wild-type or dominant negative forms of CREB and c-Fos or an activated...... form of the MAPK kinase, MEK2. Alternatively, PC12-E2 cells were treated with arachidonic acid, the cAMP analogue dBcAMP, or protein kinase A (PKA) inhibitors. The negative forms of CREB and c-Fos inhibited neurite outgrowth mediated by NCAM, arachidonic acid, dBcAMP, or MEK2. Neither CREB nor c...

Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia.

Science.gov (United States)

Yeow, Tee Cian; Wong, Won Fen; Sabet, Negar Shafiei; Sulaiman, Sofiah; Shahhosseini, Fatemeh; Tan, Grace Min Yi; Movahed, Elaheh; Looi, Chung Yeng; Shankar, Esaki M; Gupta, Rishien; Arulanandam, Bernard P; Hassan, Jamiyah; Abu Bakar, Sazaly

2016-03-18

The 7.5 kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis-infected patients. A total of 180 female patients of child bearing age (mean 30.9 years old, IQR:27-35) with gynecological complications and subfertility issues, who visited Obstetrics and Gynecology clinics in Kuala Lumpur, Malaysia were recruited for the study. Prevalence of genital chlamydial infection among these patients was alarmingly high at 51.1% (92/180). Of the 92 chlamydia-infected patients, 93.5% (86/92) were infected with plasmid-bearing (+) C. trachomatis while the remaining 6.5% (6/92) were caused by the plasmid-free (-) variant. Our data showed that genital C. trachomatis infection was associated with infertility issues, inflammation in the reproductive tract (mucopurulent cervicitis or endometriosis), irregular menstrual cycles and polycystic ovarian syndrome (PCOS). However, no statistical significance was detected among patients with plasmid (+) versus plasmid (-) C. trachomatis infection. Interestingly, plasmid (+) C. trachomatis was detected in all patients with PCOS, and the plasmid copy numbers were significantly higher among PCOS patients, relative to non-PCOS patients. Our findings show a high incidence of C. trachomatis infection among women with infertility or gynecological problems in Malaysia. However, due to the low number of plasmid (-) C. trachomatis cases, a significant role of the plasmid in causing virulence in human requires further investigation of a larger cohort.

Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme

NARCIS (Netherlands)

van der Ende, A.; Baker, T. A.; Ogawa, T.; Kornberg, A.

1985-01-01

The enzymatic replication of plasmids containing the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme priming systems: primase alone, RNA polymerase alone, or both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Kornberg, A.

24 CFR 221.790 - Debenture interest rate.

Science.gov (United States)

2010-04-01

... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Debenture interest rate. 221.790...-Moderate Income Projects § 221.790 Debenture interest rate. The debentures issued pursuant to the exercise of an assignment option shall bear interest at the going Federal rate at date of issuance. The going...

24 CFR 221.254 - Mortgage insurance premiums.

Science.gov (United States)

2010-04-01

... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Mortgage insurance premiums. 221... Cost Homes § 221.254 Mortgage insurance premiums. (a) All of the provisions of §§ 203.260 through 203.295 of this chapter relating to mortgage insurance premiums shall apply to mortgages insured under...

Initiation and termination of the bacteriophage phi X174 rolling circle DNA replication in vivo: packaging of plasmid single-stranded DNA into bacteriophage phi X174 coats

NARCIS (Netherlands)

van der Ende, A.; Teertstra, R.; Weisbeek, P. J.

1982-01-01

The bacteriophage phi X174 viral (+) origin when inserted in a plasmid can interact in vivo with the A protein produced by infecting phi X174 phages. A consequence of this interaction is packaging of single-stranded plasmid DNA into preformed phage coats resulting in infective particles (1). This

Plasmid fermentation process for DNA immunization applications.

Science.gov (United States)

Carnes, Aaron E; Williams, James A

2014-01-01

Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. This chapter describes a simple fed-batch fermentation process for producing bacterial cell paste enriched with high-quality plasmid DNA. A constant feeding strategy results in a medium cell density culture with continuously increasing plasmid amplification towards the end of the process. Cell banking and seed culture preparation protocols, which can dramatically influence final product yield and quality, are also described. These protocols are suitable for production of research-grade plasmid DNA at the 100 mg-to-1.5 g scale from a typical 10 L laboratory benchtop fermentor.

Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family).

Science.gov (United States)

Kulinska, Anna; Godziszewska, Jolanta; Wojciechowska, Anna; Ludwiczak, Marta; Jagura-Burdzy, Grazyna

2016-04-01

The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parSRA3, forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nicoperon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions in Escherichia coli, since its overexpression in transnegatively interferes with mini-RA3 replication and stable maintenance of RA3. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

39 CFR 221.7 - Postal Service emblem.

Science.gov (United States)

2010-07-01

... 39 Postal Service 1 2010-07-01 2010-07-01 false Postal Service emblem. 221.7 Section 221.7 Postal... Service emblem. The Postal Service emblem, which is identical with the seal, is registered as a trademark and service mark by the U.S. Patent Office. Except for the emblem on official stationery, the emblem...

Antimicrobial susceptibility pattern and plasmid-mediated ...

African Journals Online (AJOL)

negative Staphylococci (CoNS) were isolated from clinical samples and isolates subjected to antibiotic susceptibility testing, plasmid curing and plasmid DNA isolation. Result: The highest percentages isolates were recovered from urine samples and ...

46 CFR 221.111 - Status of prior transactions-controlling dates.

Science.gov (United States)

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Status of prior transactions-controlling dates. 221.111 Section 221.111 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION REGULATIONS AFFECTING... MARITIME INTERESTS Savings Provisions § 221.111 Status of prior transactions—controlling dates. (a) The...

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

24 CFR 221.795 - Displacement-below market interest rate mortgages.

Science.gov (United States)

2010-04-01

... Rights and Obligations-Moderate Income Projects § 221.795 Displacement—below market interest rate... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Displacement-below market interest rate mortgages. 221.795 Section 221.795 Housing and Urban Development Regulations Relating to Housing...

50 CFR 221.4 - What deadlines apply to pending applications?

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 7 2010-10-01 2010-10-01 false What deadlines apply to pending applications? 221.4 Section 221.4 Wildlife and Fisheries NATIONAL MARINE FISHERIES SERVICE, NATIONAL OCEANIC... LICENSES General Provisions § 221.4 What deadlines apply to pending applications? (a) Applicability. (1...

PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1.

Science.gov (United States)

Vogt, Julia; Wernstedt, Annekatrin; Ripperger, Tim; Pabst, Brigitte; Zschocke, Johannes; Kratz, Christian; Wimmer, Katharina

2016-11-01

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5'-part from the 3'-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms.

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

Science.gov (United States)

Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L

2012-01-01

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

Directory of Open Access Journals (Sweden)

Mark Eppinger

Full Text Available Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

14 CFR 221.102 - Accessibility of tariffs to the public.

Science.gov (United States)

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Accessibility of tariffs to the public. 221.102 Section 221.102 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION... Inspection § 221.102 Accessibility of tariffs to the public. Each file of tariffs shall be kept in complete...

Theragnosis-based combined cancer therapy using doxorubicin-conjugated microRNA-221 molecular beacon.

Science.gov (United States)

Lee, Jonghwan; Choi, Kyung-Ju; Moon, Sung Ung; Kim, Soonhag

2016-01-01

Recently, microRNA (miRNA or miR) has emerged as a new cancer biomarker because of its high expression level in various cancer types and its role in the control of tumor suppressor genes. In cancer studies, molecular imaging and treatment based on target cancer markers have been combined to facilitate simultaneous cancer diagnosis and therapy. In this study, for combined therapy with diagnosis of cancer, we developed a doxorubicin-conjugated miR-221 molecular beacon (miR-221 DOXO MB) in a single platform composed of three different nucleotides: miR-221 binding sequence, black hole quencher 1 (BHQ1), and doxorubicin binding site. Imaging of endogenous miR-221 was achieved by specific hybridization between miR-221 and the miR-221 binding site in miR-221 DOXO MB. The presence of miR-221 triggered detachment of the quencher oligo and subsequent activation of a fluorescent signal of miR-221 DOXO MB. Simultaneous cancer therapy in C6 astrocytoma cells and nude mice was achieved by inhibition of miRNA-221 function that downregulates tumor suppressor genes. The detection of miR-221 expression and inhibition of miR-221 function by miR-221 DOXO MB provide the feasibility as a cancer theragnostic probe. Furthermore, a cytotoxic effect was induced by unloading of doxorubicin intercalated into miR-221 DOXO MB inside cells. Loss of miR-221 function and cytotoxicity induced by the miR-221 DOXO MB provides combined therapeutic efficacy against cancers. This method could be used as a new theragnostic probe with enhanced therapy to detect and inhibit many cancer-related miRNAs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of a Staphylococcal Plasmid Related to pUB110 and Carrying Two Novel Genes, vatC and vgbB, Encoding Resistance to Streptogramins A and B and Similar Antibiotics

Science.gov (United States)

Allignet, Jeanine; Liassine, Nadia; El Solh, Névine

1998-01-01

We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation. PMID:9661023

Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and B and similar antibiotics.

Science.gov (United States)

Allignet, J; Liassine, N; el Solh, N

1998-07-01

We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation.

Multilocus sequence typing of IncN plasmids

DEFF Research Database (Denmark)

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

2011-01-01

that spread and persistence of this particular IncN-carrying blaVIM-1 lineage in Greece. CONCLUSIONS: This study proposes the use of pMLST as a suitable and rapid method for identification of IncN epidemic plasmid lineages. The recent spread of blaCTX-M-1 among humans and animals seems to be associated......OBJECTIVES: Incompatibility group N (IncN) plasmids have been associated with the dissemination of antimicrobial resistance and are a major vehicle for the spread of blaVIM-1 in humans and blaCTX-M-1 in animals. A plasmid multilocus sequence typing (pMLST) scheme was developed for rapid...... in different countries from both animals and humans belonged to ST1, suggesting dissemination of an epidemic plasmid through the food chain. Fifteen of 17 plasmids carrying blaVIM-1 from Klebsiella pneumoniae and Escherichia coli, isolated during a 5year period in Greece were assigned to ST10, suggesting...

A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

Science.gov (United States)

Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

2016-01-01

Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

76 FR 5467 - Airworthiness Directives; Pilatus Aircraft Ltd. Models PC-6, PC-6-H1, PC-6-H2, PC-6/350, PC-6/350...

Science.gov (United States)

2011-02-01

...;Prices of new books are listed in the first FEDERAL REGISTER issue of each #0;week. #0; #0; #0; #0;#0..., Fairchild Heli Porter PC-6 airplanes, or Fairchild-Hiller Corporation PC-6 airplanes. Discussion Section... [[Page 5469

24 CFR 221.256 - Interest rate increase and payment of mortgage insurance premiums on mortgages under § 221.60 and...

Science.gov (United States)

2010-04-01

... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Interest rate increase and payment... Interest rate increase and payment of mortgage insurance premiums on mortgages under § 221.60 and § 221.65... continuation of a below market interest rate, interest on such mortgage shall be computed by the mortgagee at...

Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.

Directory of Open Access Journals (Sweden)

Zihua Wang

Full Text Available Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV, a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg RNA which is also required as bicistronic mRNA for the capsid (core protein and the reverse transcriptase (Pol; their open reading frames (ORFs overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES. We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR and humanized Renilla green fluorescent protein (hrGFP produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to

Compatibility and entry exclusion of IncA and IncC plasmids revisited: IncA and IncC plasmids are compatible.

Science.gov (United States)

Ambrose, Stephanie J; Harmer, Christopher J; Hall, Ruth M

2018-02-24

In an early study, IncA and IncC plasmids that were reported to be compatible were grouped as the "A-C complex" based on similarities and on strong entry exclusion. However, recently, the term IncA/C has been used frequently to describe plasmids belonging to both of these two groups. Granted that the supporting data was not included in the original reports and that the consensus iteron sequences have since been shown to be essentially identical, we have addressed the question again. The original IncA plasmid, RA1, and the IncC plasmid pRMH760, were introduced into the same cell by transformation, and were found to be maintained stably for over 100 generations in the absence of selection for either plasmid, i.e. they were compatible. We conclude that use of the term IncA/C for this important plasmid group is indeed incorrect and it causes unnecessary confusion. Granted the importance of IncC plasmids in the spread of antibiotic resistance genes, we recommend that use of the misleading terms IncA/C, IncA/C 1 and IncA/C 2 should cease. In addition, RA1 and pRMH760 were shown to each completely prevent entry of the other via conjugative transfer into the cell they reside in. Copyright © 2018 Elsevier Inc. All rights reserved.

Ecological and genetic determinants of plasmid distribution in Escherichia coli.

Science.gov (United States)

Medaney, Frances; Ellis, Richard J; Raymond, Ben

2016-11-01

Bacterial plasmids are important carriers of virulence and antibiotic resistance genes. Nevertheless, little is known of the determinants of plasmid distribution in bacterial populations. Here the factors affecting the diversity and distribution of the large plasmids of Escherichia coli were explored in cattle grazing on semi-natural grassland, a set of populations with low frequencies of antibiotic resistance genes. Critically, the population genetic structure of bacterial hosts was chararacterized. This revealed structured E. coli populations with high diversity between sites and individuals but low diversity within cattle hosts. Plasmid profiles, however, varied considerably within the same E. coli genotype. Both ecological and genetic factors affected plasmid distribution: plasmid profiles were affected by site, E. coli diversity, E. coli genotype and the presence of other large plasmids. Notably 3/26 E. coli serotypes accounted for half the observed plasmid-free isolates indicating that within species variation can substantially affect carriage of the major conjugative plasmids. The observed population structure suggest that most of the opportunities for within species plasmid transfer occur between different individuals of the same genotype and support recent experimental work indicating that plasmid-host coevolution, and epistatic interactions on fitness costs are likely to be important in determining occupancy. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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.

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

Directory of Open Access Journals (Sweden)

Caroline M Li

Full Text Available 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.

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.

The characteristics of micrococcus (deinococcus) radiodurans sark plasmids

International Nuclear Information System (INIS)

Sjarief, Sri Hariani; Kikuchi, Masahiro; Watanabe, Hiroshi.

1994-01-01

The characterization of micrococcus (deinococcus) radiodurans sark plasmids. This bacterium has been classified as a new genus deinococcus radiodurans which is resistant to gamma-rays. It can repair itself completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 KGy. To reveal the repair mechanism, several investigations had been done to develop a cloning vector available for the genetic analysis. For this purpose D. radiodurans Sark are to be prepared as a vector by studying the characteristics of its plasmid. Plasmids were isolated by electrophoresis using 0.6% low-melting-temperature agarose in TAE and run for 5.5 hours, followed by the identification. An antibiotic marker was also carried out in this experiment to identify its location in the genetic materials of the cell, beside making a restriction map of the plasmid. Results have shown that D. radiodurans Sark has 4 plasmids (P1, P2, P3, and P4) and the refampicin resistant genes were not found in the plasmid. (authors). 14 refs; 4 figs

Lactose carrier protein of Escherichia coli. Structure and expression of plasmids carrying the Y gene of the lac operon.

Science.gov (United States)

Teather, R M; Bramhall, J; Riede, I; Wright, J K; Fürst, M; Aichele, G; Wilhelm, U; Overath, P

1980-01-01

The previously described hybrid plasmid pC7 which carries lacI+O+delta(Z)Y+A+ on a 12.3 X 10(6)-Mr DNA fragment [Teather et al. (1978) Mol. Gen. Genet. 159, 239-248] was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to d(A-A-T-T) and ligated to the vector pB322. lac Y-carrying inserts of various sized (Mr 1.5-4.7 X 10(6)) were obtained. Hybrid plasmid pTE18 (2300-base-pair insert) carries part of the I (repressor) gene, the promotor-operator region, part of the Z (beta-galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18-harbouring strains the Y-gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12-16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.

27 CFR 41.221 - Change in trade name.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 2 2010-04-01 2010-04-01 false Change in trade name. 41.221 Section 41.221 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU... Change in trade name. Where there is a change in, or an addition or discontinuance of, a trade name used...

Plasmids foster diversification and adaptation of bacterial populations in soil.

Science.gov (United States)

Heuer, Holger; Smalla, Kornelia

2012-11-01

It is increasingly being recognized that the transfer of conjugative plasmids across species boundaries plays a vital role in the adaptability of bacterial populations in soil. There are specific driving forces and constraints of plasmid transfer within bacterial communities in soils. Plasmid-mediated genetic variation allows bacteria to respond rapidly with adaptive responses to challenges such as irregular antibiotic or metal concentrations, or opportunities such as the utilization of xenobiotic compounds. Cultivation-independent detection and capture of plasmids from soil bacteria, and complete sequencing have provided new insights into the role and ecology of plasmids. Broad host range plasmids such as those belonging to IncP-1 transfer a wealth of accessory functions which are carried by similar plasmid backbones. Plasmids with a narrower host range can be more specifically adapted to particular species and often transfer genes which complement chromosomally encoded functions. Plasmids seem to be an ancient and successful strategy to ensure survival of a soil population in spatial and temporal heterogeneous conditions with various environmental stresses or opportunities that occur irregularly or as a novel challenge in soil. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Glycosaminoglycan composition of PC12 pheochromocytoma cells: a comparison with PC12D cells, a new subline of PC12 cells

Energy Technology Data Exchange (ETDEWEB)

Katoh-Semba, R.; Oohira, A.; Sano, M.; Watanabe, K.; Kitajima, S.; Kashiwamata, S.

1989-03-01

PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PC12D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of (/sup 35/S)sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of (/sup 35/S)sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.

VIDEO-PC, SVGA Routines for FORTRAN on PC

International Nuclear Information System (INIS)

1994-01-01

1 - Description of program or function: These primitives are the lowest level routines needed to perform super VGA graphics on a PC. A sample main program is included that exercises the primitives. Both Lahey and Microsoft FORTRAN's have graphics libraries. However, the libraries do not support 256 color graphics at resolutions greater than 320x200. The primitives bypass these libraries while still conforming to standard usage of BIOS. The supported graphics modes depend upon the PC graphics card and its memory. Super VGA resolutions of 640x480 and 800x600 have been tested on an ATI VGA Wonder card with 512 K memory and on several 80486 PC's (unknown manufacturers) at retail stores. 2 - Method of solution: Both Lahey and Microsoft primitives depend upon sending the correct parameters to the PC BIOS (basic input output system) as discussed in the references. 3 - Restrictions on the complexity of the problem: The primitives were developed for 256 color VGA applications. It is known, for instance, that some CGA and 16 color VGA video modes will not operate correctly using these primitives. Potential users should try the test program on their PC/video card combination to determine applicability

Biomimicry 1: PC.

Science.gov (United States)

Cumberland, D C; Gunn, J; Malik, N; Holt, C M

1998-01-01

The surface properties of stents can be modified by coating them, for example with a polymer. Phosphorylcoline (PC) is the major component of the outer layer of the cell membrane. The haemo- and biocompatibility of a PC-containing polymer is thus based on biomimicry, and has been confirmed by several experiments showing much reduced thrombogenicity of PC-coated surfaces, and porcine coronary artery implants showing no sign of adverse effect. Clinical experience with the PC-coated BiodivYsio appears favourable. The PC coating can be tailored for take up and controlled elution of various drugs for stent-based local delivery, a property which is being actively explored.

Effects of GCK, GCKR, G6PC2 and MTNR1B variants on glucose metabolism and insulin secretion.

Directory of Open Access Journals (Sweden)

Cheng Hu

Full Text Available BACKGROUND: Single nucleotide polymorphisms (SNPs from GCK, GCKR, G6PC2 and MTNR1B were found to modulate the fasting glucose levels. The current study aimed to replicate this association in the Chinese population and further analyze their effects on biphasic insulin secretion. METHODS/PRINCIPAL FINDINGS: SNPs from GCK, GCKR, G6PC2 and MTNR1B were genotyped in the Shanghai Chinese, including 3,410 type 2 diabetes patients and 3,412 controls. The controls were extensively phenotyped for the traits related to glucose metabolism and insulin secretion. We replicated the association between GCK rs1799884, G6PC2 rs16856187 and MTNR1B rs10830963 and fasting glucose in our samples (p = 0.0003-2.0x10(-8. GCK rs1799884 and G6PC2 rs16856187 showed association to HOMA-beta, insulinogenic index and both first- and second-phases insulin secretion (p = 0.0030-0.0396. MTNR1B rs10830963 was associated to HOMA-beta, insulinogenic index and first-phase insulin secretion (p = 0.0102-0.0426, but not second-phase insulin secretion (p = 0.9933. Combined effect analyses showed individuals carrying more risk allele for high fasting glucose tended to have a higher glucose levels at both fasting and 2 h during OGTTs (p = 1.7x10(-13 and 0.0009, respectively, as well as lower HOMA-beta, insulinogenic index and both first- and second-phases insulin secretion (p = 0.0321-1.1x10(-7. CONCLUSIONS/SIGNIFICANCE: We showed that SNPs from GCK, G6PC2 and MTNR1B modulated the fasting glucose levels in the normoglycaemic population while SNPs from G6PC2 and GCKR was associated with type 2 diabetes. Moreover, we found GCK and G6PC2 genetic variants were associated to both first- and second-phases insulin secretion while MTNR1B genetic variant was associated with first-phase insulin secretion, but not second-phase insulin secretion.

Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

Science.gov (United States)

Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

2017-11-01

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

Mutation in ESBL Plasmid from Escherichia coli O104:H4 Leads Autoagglutination and Enhanced Plasmid Dissemination

Directory of Open Access Journals (Sweden)

Mickaël Poidevin

2018-02-01

Full Text Available Conjugative plasmids are one of the main driving force of wide-spreading of multidrug resistance (MDR bacteria. They are self-transmittable via conjugation as carrying the required set of genes and cis-acting DNA locus for direct cell-to-cell transfer. IncI incompatibility plasmids are nowadays often associated with extended-spectrum beta-lactamases producing Enterobacteria in clinic and environment. pESBL-EA11 was isolated from Escherichia coli O104:H4 outbreak strain in Germany in 2011. During the previous study identifying transfer genes of pESBL-EA11, it was shown that transposon insertion at certain DNA region of the plasmid, referred to as Hft, resulted in great enhancement of transfer ability. This suggested that genetic modifications can enhance dissemination of MDR plasmids. Such ‘superspreader’ mutations have attracted little attention so far despite their high potential to worsen MDR spreading. Present study aimed to gain our understanding on regulatory elements that involved pESBL transfer. While previous studies of IncI plasmids indicated that immediate downstream gene of Hft, traA, is not essential for conjugative transfer, here we showed that overexpression of TraA in host cell elevated transfer rate of pESBL-EA11. Transposon insertion or certain nucleotide substitutions in Hft led strong TraA overexpression which resulted in activation of essential regulator TraB and likely overexpression of conjugative pili. Atmospheric Scanning Electron Microscopy observation suggested that IncI pili are distinct from other types of conjugative pili (such as long filamentous F-type pili and rather expressed throughout the cell surface. High transfer efficiency in the mutant pESBL-EA11 was involved with hyperpiliation which facilitates cell-to-cell adhesion, including autoagglutination. The capability of plasmids to evolve to highly transmissible mutant is alarming, particularly it might also have adverse effect on host pathogenicity.

Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast-derived cells.

Science.gov (United States)

Houba-Hérin, N; Domin, M; Pédron, J

1994-07-01

Nicotiana plumbaginifolia haploid protoplasts were co-transformed with two plasmids, one with a NPT-II/Ds element and one with a gene encoding an amino-terminal truncated Ac transposase. It is shown that Ds can efficiently transpose from extrachromosomal DNA to N. plumbaginifolia chromosomes when the Ac transposase gene is present in trans. Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 transgenic lines tested. The flanking sequences present in the original plasmid are missing in these 21 plants. In only two of 21 plants was part of the transposase construct integrated. By segregation analysis of transgenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplasts had been originally transformed. This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.

Cdc7 is required throughout the yeast S phase to activate replication origins.

Science.gov (United States)

Donaldson, A D; Fangman, W L; Brewer, B J

1998-02-15

The long-standing conclusion that the Cdc7 kinase of Saccharomyces cerevisiae is required only to trigger S phase has been challenged by recent data that suggests it acts directly on individual replication origins. We tested the possibility that early- and late-activated origins have different requirements for Cdc7 activity. Cells carrying a cdc7(ts) allele were first arrested in G1 at the cdc7 block by incubation at 37 degrees C, and then were allowed to enter S phase by brief incubation at 23 degrees C. During the S phase, after return to 37 degrees C, early-firing replication origins were activated, but late origins failed to fire. Similarly, a plasmid with a late-activated origin was defective in replication. As a consequence of the origin activation defect, duplication of chromosomal sequences that are normally replicated from late origins was greatly delayed. Early-replicating regions of the genome duplicated at approximately their normal time. The requirements of early and late origins for Cdc7 appear to be temporally rather than quantitatively different, as reducing overall levels of Cdc7 by growth at semi-permissive temperature reduced activation at early and late origins approximately equally. Our results show that Cdc7 activates early and late origins separately, with late origins requiring the activity later in S phase to permit replication initiation.

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

International Nuclear Information System (INIS)

Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I.

1990-01-01

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores

Data Quality Objectives Summary Report for the 221-U Canyon Disposition Alternatives

International Nuclear Information System (INIS)

Miller, M.S.; Oaces, L.E.; Baxter, J.; Brown, T.M.; Enoke, D.E.; Carlson, D.; Rugg, J.E.

1997-08-01

The 221-U Canyon Disposition Alternatives Data Quality Objective (DQO) Process identifies the sampling and analytical requirements necessary to support future detailed evaluation of alternatives via the CERCLA process, for final disposition of the 221-U Canyon Facility. Viable alternatives for the disposition of the 221-U Facility have been identified in a CERCLA Phase I Feasibility Study (FS) (DOE-RL 1997) for the Canyon Disposition Initiative (CDI). The scope of this DQO Process is limited to the 221-U Process Canyon Building and equipment contained within the facility. Associated stacks, filters, solvent handling, vaults, and storage facilities external to the 221-U Building are not addressed in this DQO. This DQO focuses on the 221-U Building because it provides the greatest potential source of contaminant volumes and concentrations and the physical structure poses the greatest challenge for disposition decisions

Strong minor groove base conservation in sequence logos implies DNA distortion or base flipping during replication and transcription initiation | Center for Cancer Research

Science.gov (United States)

Dubbed "Tom's T" by Dhruba Chattoraj, the unusually conserved thymine at position +7 in bacteriophage P1 plasmid RepA DNA binding sites rises above repressor and acceptor sequence logos. The T appears to represent base flipping prior to helix opening in this DNA replication initation protein.

20 CFR 404.221 - Computing your average monthly wage.

Science.gov (United States)

2010-04-01

... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Computing your average monthly wage. 404.221... DISABILITY INSURANCE (1950- ) Computing Primary Insurance Amounts Average-Monthly-Wage Method of Computing Primary Insurance Amounts § 404.221 Computing your average monthly wage. (a) General. Under the average...

Duck enteritis virus glycoprotein D and B DNA vaccines induce immune responses and immunoprotection in Pekin ducks.

Science.gov (United States)

Zhao, Yan; Cao, Yongsheng; Cui, Lihong; Ma, Bo; Mu, Xiaoyu; Li, Yanwei; Zhang, Zhihui; Li, Dan; Wei, Wei; Gao, Mingchun; Wang, Junwei

2014-01-01

DNA vaccine is a promising strategy for protection against virus infection. However, little is known on the efficacy of vaccination with two plasmids for expressing the glycoprotein D (gD) and glycoprotein B (gB) of duck enteritis virus (DEV) in inducing immune response and immunoprotection against virulent virus infection in Pekin ducks. In this study, two eukaryotic expressing plasmids of pcDNA3.1-gB and pcDNA3.1-gD were constructed. Following transfection, the gB and gD expressions in DF1 cells were detected. Groups of ducks were vaccinated with pcDNA3.1-gB and/or pcDNA3.1-gD, and boosted with the same vaccine on day 14 post primary vaccination. We found that intramuscular vaccinations with pcDNA3.1-gB and/or pcDNA3.1-gD, but not control plasmid, stimulated a high frequency of CD4+ and CD8+ T cells in Pekin ducks, particularly with both plasmids. Similarly, vaccination with these plasmids, particularly with both plasmids, promoted higher levels of neutralization antibodies against DEV in Pekin ducks. More importantly, vaccination with both plasmids significantly reduced the virulent DEV-induced mortality in Pekin ducks. Our data indicated that vaccination with plasmids for expressing both gB and gD induced potent cellular and humoral immunity against DEV in Pekin ducks. Therefore, this vaccination strategy may be used for the prevention of DEV infection in Pekin ducks.

Analysis of the temporal program of replication initiation in yeast chromosomes.

Science.gov (United States)

Friedman, K L; Raghuraman, M K; Fangman, W L; Brewer, B J

1995-01-01

The multiple origins of eukaryotic chromosomes vary in the time of their initiation during S phase. In the chromosomes of Saccharomyces cerevisiae the presence of a functional telomere causes nearby origins to delay initiation until the second half of S phase. The key feature of telomeres that causes the replication delay is the telomeric sequence (C(1-3)A/G(1-3)T) itself and not the proximity of the origin to a DNA end. A second group of late replicating origins has been found at an internal position on chromosome XIV. Four origins, spanning approximately 140 kb, initiate replication in the second half of S phase. At least two of these internal origins maintain their late replication time on circular plasmids. Each of these origins can be separated into two functional elements: those sequences that provide origin function and those that impose late activation. Because the assay for determining replication time is costly and laborious, it has not been possible to analyze in detail these 'late' elements. We report here the development of two new assays for determining replication time. The first exploits the expression of the Escherichia coli dam methylase in yeast and the characteristic period of hemimethylation that transiently follows the passage of a replication fork. The second uses quantitative hybridization to detect two-fold differences in the amount of specific restriction fragments as a function of progress through S phase. The novel aspect of this assay is the creation in vivo of a non-replicating DNA sequence by site-specific pop-out recombination. This non-replicating fragment acts as an internal control for copy number within and between samples. Both of these techniques are rapid and much less costly than the more conventional density transfer experiments that require CsCl gradients to detect replicated DNA. With these techniques it should be possible to identify the sequences responsible for late initiation, to search for other late replicating

Plasmid-Mediated Antimicrobial Resistance in Staphylococci and Other Firmicutes.

Science.gov (United States)

Schwarz, Stefan; Shen, Jianzhong; Wendlandt, Sarah; Fessler, Andrea T; Wang, Yang; Kadlec, Kristina; Wu, Cong-Ming

2014-12-01

In staphylococci and other Firmicutes, resistance to numerous classes of antimicrobial agents, which are commonly used in human and veterinary medicine, is mediated by genes that are associated with mobile genetic elements. The gene products of some of these antimicrobial resistance genes confer resistance to only specific members of a certain class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into any of three major categories: active efflux, enzymatic inactivation, and modification/replacement/protection of the target sites of the antimicrobial agents. Among the mobile genetic elements that carry such resistance genes, plasmids play an important role as carriers of primarily plasmid-borne resistance genes, but also as vectors for nonconjugative and conjugative transposons that harbor resistance genes. Plasmids can be exchanged by horizontal gene transfer between members of the same species but also between bacteria belonging to different species and genera. Plasmids are highly flexible elements, and various mechanisms exist by which plasmids can recombine, form cointegrates, or become integrated in part or in toto into the chromosomal DNA or into other plasmids. As such, plasmids play a key role in the dissemination of antimicrobial resistance genes within the gene pool to which staphylococci and other Firmicutes have access. This chapter is intended to provide an overview of the current knowledge of plasmid-mediated antimicrobial resistance in staphylococci and other Firmicutes.

Convergent evolution of Amadori opine catabolic systems in plasmids of Agrobacterium tumefaciens.

Science.gov (United States)

Baek, Chang-Ho; Farrand, Stephen K; Lee, Ko-Eun; Park, Dae-Kyun; Lee, Jeong Kug; Kim, Kun-Soo

2003-01-01

Deoxyfructosyl glutamine (DFG, referred to elsewhere as dfg) is a naturally occurring Amadori compound found in rotting fruits and vegetables. DFG also is an opine and is found in tumors induced by chrysopine-type strains of Agrobacterium tumefaciens. Such strains catabolize this opine via a pathway coded for by their plasmids. NT1, a derivative of the nopaline-type A. tumefaciens strain C58 lacking pTiC58, can utilize DFG as the sole carbon source. Genes for utilization of DFG were mapped to the 543-kb accessory plasmid pAtC58. Two cosmid clones of pAtC58 allowed UIA5, a plasmid-free derivative of C58, harboring pSa-C that expresses MocC (mannopine [MOP] oxidoreductase that oxidizes MOP to DFG), to grow by using MOP as the sole carbon source. Genetic analysis of subclones indicated that the genes for utilization of DFG are located in a 6.2-kb BglII (Bg2) region adjacent to repABC-type genes probably responsible for the replication of pAtC58. This region contains five open reading frames organized into at least two transcriptional soc (santhopine catabolism) groups: socR and socABCD. Nucleotide sequence analysis and analyses of transposon-insertion mutations in the region showed that SocR negatively regulates the expression of socR itself and socABCD. SocA and SocB are responsible for transport of DFG and MOP. SocA is a homolog of known periplasmic amino acid binding proteins. The N-terminal half of SocB is a homolog of the transmembrane transporter proteins for several amino acids, and the C-terminal half is a homolog of the transporter-associated ATP-binding proteins. SocC and SocD could be responsible for the enzymatic degradation of DFG, being homologs of sugar oxidoreductases and an amadoriase from Corynebacterium sp., respectively. The protein products of socABCD are not related at the amino acid sequence level to those of the moc and mot genes of Ti plasmids responsible for utilization of DFG and MOP, indicating that these two sets of genes and their

Adenovirus or HA-2 fusogenic peptide-assisted lipofection increases cytoplasmic levels of plasmid in nondividing endothelium with little enhancement of transgene expression.

Science.gov (United States)

Subramanian, Ajit; Ma, Haiching; Dahl, Kris N; Zhu, Jingya; Diamond, Scott L

2002-01-01

Adenovirus-assisted lipofection has been reported to increase transfection efficiency through mechanisms potentially involving endosome escape and/or nuclear targeting activity. Similarly, transfection with the viral fusogenic peptide HA-2 of the influenza virus hemagglutinin can increase transfection efficiency. However, there are few studies examining the mechanism and intracellular trafficking of these viral and/or viral fusogenic peptide-assisted lipofections. Endosome escape was directly assayed with T7 RNA polymerase bound to plasmid (pTM beta gal) expressing beta-galactosidase under a T7 promoter to detect transcribable plasmid that escapes the endosomal compartment. Lipofection of pTM beta gal with replication-deficient adenovirus (Ad5-null) at a multiplicity of infection (MOI) of 100 and 1000 increased cytoplasmic levels of transcribable plasmid by 24- and 117-fold, respectively, over lipofection alone, without an effect on total plasmid uptake. However, lipofection of pCMV beta gal with Ad5-null at a MOI of 100 and 1000 increased transgene expression only seven- and eight-fold, respectively, over lipofection alone. Thus, a 24-fold increase in endosome escape saturated expression from pCMV beta gal and provided only a seven-fold benefit in nondividing cells, which was not significantly increased with further increases in endosome escape. A cationic form of HA-2 (HA-K(4)) also caused significant enhancements in endosome escape, as detected with the cytoplasmic transcription assay. However, HA-K(4) enhancement of endosome escape did not correlate with transgene expression from pCMV beta gal, consistent with the detection of HA-K(4)-mediated partitioning of plasmid to the insoluble fraction of the cell lysate. These results indicate that enhancement of endosome escape in nondividing cells does not fully alleviate rate limits related to nuclear import of the plasmid. Copyright 2001 John Wiley & Sons, Ltd.

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity

DEFF Research Database (Denmark)

Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno

2004-01-01

or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone......, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX......[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570...

Comparative genomics of the IncA/C multidrug resistance plasmid family.

Science.gov (United States)

Fricke, W Florian; Welch, Timothy J; McDermott, Patrick F; Mammel, Mark K; LeClerc, J Eugene; White, David G; Cebula, Thomas A; Ravel, Jacques

2009-08-01

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

12 CFR 221.125 - Credit to brokers and dealers.

Science.gov (United States)

2010-01-01

... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Credit to brokers and dealers. 221.125 Section... SYSTEM CREDIT BY BANKS AND PERSONS OTHER THAN BROKERS OR DEALERS FOR THE PURPOSE OF PURCHASING OR CARRYING MARGIN STOCK (REGULATION U) Interpretations § 221.125 Credit to brokers and dealers. (a) The...

12 CFR 221.103 - Loans to brokers or dealers.

Science.gov (United States)

2010-01-01

... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Loans to brokers or dealers. 221.103 Section... SYSTEM CREDIT BY BANKS AND PERSONS OTHER THAN BROKERS OR DEALERS FOR THE PURPOSE OF PURCHASING OR CARRYING MARGIN STOCK (REGULATION U) Interpretations § 221.103 Loans to brokers or dealers. Questions have...

Plasmid mediated enhancement of uv resistance in Streptococcus faecalis

International Nuclear Information System (INIS)

Miehl, R.; Miller, M.; Yasbin, R.E.

1980-01-01

A 38.5-Mdal plasmid of Streptococcus faecalis subdp. zymogenes has been shown to enhance survival following uv irradiation. In addition, the presence of this plasmid increases the mutation frequencies following uv irradiation and enhanced W-reactivation. The data presented indicate that S. faecalis has an inducible error-prone repair system and that the plasmid enhances these repair functions

Complete Sequence of p07-406, a 24,179-base-pair plasmid harboring the blaVIM-7 metallo-beta-lactamase gene in a Pseudomonas aeruginosa isolate from the United States.

Science.gov (United States)

Li, Hongyang; Toleman, Mark A; Bennett, Peter M; Jones, Ronald N; Walsh, Timothy R

2008-09-01

An outbreak involving a Pseudomonas aeruginosa strain that was resistant to all tested antimicrobials except polymyxin B occurred in a hospital in Houston, TX. Previous studies on this strain showed that it possesses a novel mobile metallo-beta-lactamase (MBL) gene, designated bla(VIM-7), located on a plasmid (p07-406). Here, we report the complete sequence, annotation, and functional characterization of this plasmid. p07-406 is 24,179 bp in length, and 29 open reading frames were identified related to known or putatively recognized proteins. Analysis of this plasmid showed it to be comprised of four distinct regions: (i) a region of 5,200 bp having a Tn501-like mercuric resistance (mer) transposon upstream of the replication region; (ii) a Tn3-like transposon carrying a truncated integron with a bla(VIM-7) gene and an insertion sequence inserted at the other end of this transposon; (iii) a region of four genes, upstream of the Tn3-like transposon, possessing very high similarity to plasmid pXcB from Xanthomonas campestris pv. citri commonly associated with plants; (iv) a backbone sequence similar to the backbone structure of the IncP group plasmid Rms149, pB10, and R751. This is the first plasmid to be sequenced carrying an MBL gene and highlights the amelioration of DNA segments from disparate origins, most noticeably from plant pathogens.

Multiple drug resistant carbapenemases producing Acinetobacter baumannii isolates harbours multiple R-plasmids

Directory of Open Access Journals (Sweden)

Rajagopalan Saranathan

2014-01-01

Full Text Available Background & objectives: The nosocomial human pathogen Acinetobacter baumannii has high propensity to develop resistance to antimicrobials and to become multidrug resistant (MDR, consequently complicating the treatment. This study was carried out to investigate the presence of resistant plasmids (R-plasmids among the clinical isolates of A. baumannii. In addition, the study was performed to check the presence of common β-lactamases encoding genes on these plasmids. Methods: A total of 55 clinical isolates of A. baumannii were included in the study and all were subjected to plasmid DNA isolation, followed by PCR to check the presence of resistance gene determinants such as blaOXA-23 , blaOXA-51, blaOXA-58 and blaIMP-1 on these plasmids that encode for oxacillinase (OXA and metallo-β-lactamase (MBL type of carbapenemases. Plasmid curing experiments were carried out on selected isolates using ethidium bromide and acridine orange as curing agents and the antibiotic resistance profiles were evaluated before and after curing. Results: All the isolates were identified as A. baumannii by 16SrDNA amplification and sequencing. Plasmid DNA isolated from these isolates showed the occurrence of multiple plasmids with size ranging from 500bp to ≥ 25 kb. The percentage of blaOXA-51 and blaOXA-23 on plasmids were found to be 78 and 42 per cent, respectively and 20 isolates (36% carried blaIMP-1 gene on plasmids. Significant difference was observed in the antibiograms of plasmid cured isolates when compared to their parental ones. The clinical isolates became susceptible to more than two antibiotic classes after curing of plasmids indicating plasmid borne resistance. Interpretation & conclusions: Our study determined the plasmid mediated resistance mechanisms and occurrence of different resistance genes on various plasmids isolated from MDR A. baumannii. The present findings showed the evidence for antibiotic resistance mediated through multiple plasmids in

Movement and equipositioning of plasmids by ParA filament disassembly

DEFF Research Database (Denmark)

Ringgaard, Simon; van Zon, Jeroen; Howard, Martin

2009-01-01

, plasmids consistently migrate behind disassembling ParA cytoskeletal structures, suggesting that ParA filaments pull plasmids by depolymerization. The perpetual cycles of ParA assembly and disassembly result in continuous relocation of plasmids, which, on time averaging, results in equidistribution...

Neurotoxic Antibodies against the Prion Protein Do Not Trigger Prion Replication.

Directory of Open Access Journals (Sweden)

Karl Frontzek

Full Text Available Prions are the infectious agents causing transmissible spongiform encephalopathies (TSE, progressive, inexorably lethal neurological diseases. Antibodies targeting the globular domain (GD of the cellular prion protein PrPC trigger a neurotoxic syndrome morphologically and molecularly similar to prion disease. This phenomenon raises the question whether such antibodies induce infectious prions de novo. Here we exposed cerebellar organotypic cultured slices (COCS to the neurotoxic antibody, POM1. We then inoculated COCS homogenates into tga20 mice, which overexpress PrPC and are commonly utilized as sensitive indicators of prion infectivity. None of the mice inoculated with COCS-derived lysates developed any signs of disease, and all mice survived for at least 200 days post-inoculation. In contrast, all mice inoculated with bona fide prions succumbed to TSE after 55-95 days. Post-mortem analyses did not reveal any signs of prion pathology in mice inoculated with POM1-COCS lysates. Also, lysates from POM1-exposed COCS were unable to convert PrP by quaking. Hence, anti-GD antibodies do not catalyze the generation of prion infectivity. These data indicate that prion replication can be separated from prion toxicity, and suggest that anti-GD antibodies exert toxicity by acting downstream of prion replication.

Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

Science.gov (United States)

Takai, Shinji; Shoda, Masato; Sasaki, Yukako; Tsubaki, Shiro; Fortier, Guillaume; Pronost, Stephane; Rahal, Karim; Becu, Teotimo; Begg, Angela; Browning, Glenn; Nicholson, Vivian M.; Prescott, John F.

1999-01-01

Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738–740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world. PMID:10488224

Plasmid DNA Delivery: Nanotopography Matters.

Science.gov (United States)

Song, Hao; Yu, Meihua; Lu, Yao; Gu, Zhengying; Yang, Yannan; Zhang, Min; Fu, Jianye; Yu, Chengzhong

2017-12-20

Plasmid DNA molecules with unique loop structures have widespread bioapplications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry-, and flower-like morphologies composed of spike-, hemisphere-, and bowl-type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than those reported for silica-based nanovectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of nonviral vectors for efficient gene delivery.

Plasmid origin of replication of herpesvirus papio: DNA sequence and enhancer function.

Science.gov (United States)

Loeb, D D; Sung, N S; Pesano, R L; Sexton, C J; Hutchison, C; Pagano, J S

1990-01-01

Herpesvirus papio (HVP) is a lymphotropic virus of baboons which is related to Epstein-Barr virus (EBV) and produces latent infection. The nucleotide sequence of the 5,775-base-pair (bp) EcoRI K fragment of HVP, which has previously been shown to confer the ability to replicate autonomously, has been determined. Within this DNA fragment is a region which bears structural and sequence similarity to the ori-P region of EBV. The HVP ori-P region has a 10- by 26-bp tandem array which is related to the 20- by 30-bp tandem array from the EBV ori-P region. In HVP there is an intervening region of 764 bp followed by five partial copies of the 26-bp monomer. Both the EBV and HVP 3' regions have the potential to form dyad structures which, however, differ in arrangement. We also demonstrate that a transcriptional enhancer which requires transactivation by a virus-encoded factor is present in the HVP ori-P. Images PMID:2159548

DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

Directory of Open Access Journals (Sweden)

Jing Han

Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

Simple method for identification of plasmid-coded proteins

International Nuclear Information System (INIS)

Sancar, A.; Hack, A.M.; Rupp, W.D.

1979-01-01

Proteins encoded by plasmid DNA are specifically labeled in uv-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA

Radioautographic test for genetic cotton transformation by pCaVItoxneo hybrid plasmid

International Nuclear Information System (INIS)

Imamkhodjaeva, A.S.

2006-01-01

Full text: Search for novel technologies in biology, application of up-to-date methods in gene engineering, manipulation with the recombinant DNA, in particular, open opportunities for experiments with plants. To identify some DNA fragments in an organism's genome, radioautographic methods, such as dot- and blot-hybridization are frequently used. As a rule, genomic DNA is first isolated from the plant's organ. Its purification and subsequent manipulation is followed by hybridization with a probe labeled with radioactive components. The purified DNA, cDNA of RNA reverse transcription or a DNA fragment cloned in E-coli could serve as the probe. Radioautography shows homologically hybridized fragments. We have performed express dot-hybridization analysis on hybrid plasmid transformation of G.Hirsutum L. (108F) and G. Barbadense L. (C-6037) cotton sorts. pCaVItoxneo plasmid obtained on the basis of independently replicated plasmid-like DNA of the G.Hirsutum L. (pGHm2) cotton mitochondria was used (Yusupov T., 1994). There are hybrid two-domain gene of insectotoxin and enzymatically active kanamycine - phosphotransferase in the plasmid. The whole content is controlled by the plant promoter of cauliflower mosaic virus (19 S SFMV). The plasmid in question was added to the pollen sprouting medium followed by the transfer of the suspension on the pistil stigmas of the pre-prepared cotton flowers. The seed budding as the result of the experiment were analyzed by means of dot-hybridization method. DNA probes used for radioactive hybridization were labeled by method of Fainberg and Vagelstein (1990). To perform that DNA was dissolved in Tris-EDTA (10:1), containing 10mM of Tris HCl and 1mM EDTA, denaturated at 100 d eg C for 2 minutes with subsequent addition of oligonucleotide primers and annealing. DNA synthesis in the presence of 32 P labeled dATP and dCTP (Tashkent) was performed in the reaction mixture of potassium-phosphate buffer containing 67mM of MgCl 2 , 1 mg/ml of

Application of methylation in improving plasmid transformation into Helicobacter pylori.

Science.gov (United States)

Zhao, Huilin; Xu, Linlin; Rong, Qianyu; Xu, Zheng; Ding, Yunfei; Zhang, Ying; Wu, Yulong; Li, Boqing; Ji, Xiaofei

2018-05-23

Helicobacter pylori is an important gastrointestinal pathogen. Its strains possess different levels of powerful restriction modification systems, which are significant barriers to genetic tools used for studying the role of functional genes in its pathogenesis. Methylating vectors in vitro was reported as an alternative to overcome this barrier in several bacteria. In this study we used two H. pylori-E. coli shuttle plasmids and several single/double-crossover homologous recombination gene-targeting plasmids, to test the role of methylation in H. pylori transformation. According to our results, transformants could be obtained only after shuttle plasmids were methylated before transformation. It is helpful in gene complementation and over-expression although at a low frequency. The frequency of gene-targeting transformation was also increased after methylation, especially for the single-crossover recombination plasmids, the transformants of which could only be obtained after methylation. For the double-crossover recombination targeting plasmids, the initial yield of transformants was 0.3-0.8 × 10 2 CFUs per microgram plasmid DNA. With the help of methylation, the yield was increased to 0.4-1.3 × 10 2 CFUs per microgram plasmid DNA. These results suggest that in vitro methylation can improve H. pylori transformation by different plasmids, which will benefit the pathogenic mechanism research. Copyright © 2018. Published by Elsevier B.V.

White blood cells and subtypes in HFE p.C282Y and wild-type homozygotes in the Hemochromatosis and Iron Overload Screening Study.

Science.gov (United States)

Barton, James C; Barton, J Clayborn; Acton, Ronald T

2017-03-01

The major histocompatibility complex is linked to white blood cell (WBC) and lymphocyte counts in subjects unselected for HFE genotypes. We compared age, sex, body mass index, total WBC and subtypes (neutrophils, lymphocytes, monocytes, eosinophils, basophils) (Beckman Coulter® Gen-S), transferrin saturation, and serum ferritin of HFE p.C282Y and wild-type (p.C282Y, p.H63D negative) homozygotes without acquired conditions that influence WBC counts. We performed regressions on WBC and subtypes. There were 161 p.C282Y homozygotes (45.3% men) and 221 wild-type homozygotes (40.3% men). Mean WBC of men and women and between HFE genotypes were similar. Mean lymphocytes were higher in male p.C282Y homozygotes: 1.6×10 9 /L [95% confidence interval: 1.5,1.7] vs. 1.4 [1.3,1.5], p=0.0002. Mean lymphocytes and basophils were higher in female p.C282Y homozygotes: 1.6 [1.5,1.7] vs. 1.4 [1.3,1.5], p=0.0002; and 0.065 [0.059,0.071] vs. 0.052 [0.051,0.054], p=0.0001, respectively. Transferrin saturation was associated with neutrophils (negative; p=0.0163). Age was associated with lymphocytes (negative; p=0.0003) and monocytes (positive; p<0.0001). Regressions on lymphocytes and basophils revealed positive associations with p.C282Y homozygosity (p=0.0043 and 0.0003, respectively). There were significant positive associations of neutrophils, lymphocytes, monocytes, and eosinophils. We conclude that HFE p.C282Y homozygosity is significantly associated with lymphocyte and basophil counts. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

International Nuclear Information System (INIS)

Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

1984-01-01

Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. [ 3 H]Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes

Investigation of diversity of plasmids carrying the blaTEM-52 gene

DEFF Research Database (Denmark)

Bielak, Eliza Maria; Bergenholtz, Rikke D.; Jørgensen, Mikael Skaanning

2011-01-01

OBJECTIVES: To investigate the diversity of plasmids that carry blaTEM-52 genes among Escherichia coli and Salmonella enterica originating from animals, meat products and humans. METHODS: A collection of 22 blaTEM-52-encoding plasmids was characterized by restriction fragment length polymorphism...... of self-transfer to a plasmid-free E. coli recipient. CONCLUSIONS: The blaTEM-52 gene found in humans could have been transmitted on transferable plasmids originating from animal sources. Some of the blaTEM-52 plasmids carry replicons that differ from the classical ones. Two novel replicons were detected...

50 CFR 221.3 - How are time periods computed?

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 7 2010-10-01 2010-10-01 false How are time periods computed? 221.3... Provisions § 221.3 How are time periods computed? (a) General. Time periods are computed as follows: (1) The day of the act or event from which the period begins to run is not included. (2) The last day of the...

Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727

Directory of Open Access Journals (Sweden)

Mahillon Jacques

2005-07-01

Full Text Available Abstract Background Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis belong to the genetically close-knit Bacillus cereus sensu lato group, a family of rod-shaped Gram-positive bacteria. pAW63 is the first conjugative plasmid from the B. cereus group to be completely sequenced. Results The 71,777 bp nucleotide sequence of pAW63 reveals a modular structure, including a 42 kb tra region encoding homologs of the Type IV secretion systems components VirB11, VirB4 and VirD4, as well as homologs of Gram-positive conjugation genes from Enterococcus, Lactococcus, Listeria, Streptococcus and Staphylococcus species. It also firmly establishes the existence of a common backbone between pAW63, pXO2 from Bacillus anthracis and pBT9727 from the pathogenic Bacillus thuringiensis serovar konkukian strain 97-27. The alignment of these three plasmids highlights the presence of well conserved segments, in contrast to distinct regions of high sequence plasticity. The study of their specific differences has provided a three-point reference framework that can be exploited to formulate solid hypotheses concerning the functionalities and the molecular evolution of these three closely related plasmids. This has provided insight into the chronology of their divergence, and led to the discovery of two Type II introns on pAW63, matching copies of the mobile element IS231L in different loci of pXO2 and pBT9727, and the identification on pXO2 of a 37 kb pathogenicity island (PAI containing the anthrax capsule genes. Conclusion The complete sequence determination of pAW63 has led to a functional map of the plasmid yielding insights into its conjugative apparatus, which includes T4SS-like components, as well as its resemblance to other large plasmids of Gram-positive bacteria. Of particular interest is the extensive homology shared between pAW63 and pXO2, the second virulence plasmid of B. anthracis, as well as pBT9727 from the pathogenic strain B. thuringiensis

Complete Sequence of Four Multidrug-Resistant MOBQ1 Plasmids Harboring blaGES-5 Isolated from Escherichia coli and Serratia marcescens Persisting in a Hospital in Canada.

Science.gov (United States)

Boyd, David; Taylor, Geoffrey; Fuller, Jeff; Bryce, Elizabeth; Embree, Joanne; Gravel, Denise; Katz, Kevin; Kibsey, Pamela; Kuhn, Magdalena; Langley, Joanne; Mataseje, Laura; Mitchell, Robyn; Roscoe, Diane; Simor, Andrew; Thomas, Eva; Turgeon, Nathalie; Mulvey, Michael

2015-06-01

The usefulness of carbapenems for gram-negative infections is becoming compromised by organisms harboring carbapenemases, enzymes which can hydrolyze the drug. Currently KPC (class A), NDM (class B), and OXA-48 types (class D) are the most globally widespread carbapenemases. However, among the GES-type class A extended-spectrum β-lactamases (ESBLs) there are variants that hydrolyze carbapenems, with blaGES-5 being the most common. Two Escherichia coli and two Serratia marcescens harboring blaGES-5 on plasmids were isolated by the Canadian Nosocomial Infection Surveillance Program (CNISP) from four different patients in a single hospital over a 2-year period. Complete sequencing of the blaGES-5 plasmids indicated that all four had nearly identical backbones consisting of genes for replication, partitioning, and stability, but contained variant accessory regions consisting of mobile elements and antimicrobial resistance genes. The plasmids were of a novel replicon type, but belonged to the MOBQ1 group based on relaxase sequences, and appeared to be mobilizable, but not self-transmissible. Considering the time periods of bacterial isolation, it would appear the blaGES-5 plasmid has persisted in an environmental niche for at least 2 years in the hospital. This has implications for infection control and clinical care when it is transferred to clinically relevant gram-negative organisms.

Permissiveness of soil microbial communities towards broad host range plasmids

DEFF Research Database (Denmark)

Klümper, Uli

. Plasmids are implicated in the rapid spread of antibiotic resistance and the emergence of multi-resistant pathogenic bacteria, making it crucial to be able to quantify, understand, and, ideally, control plasmid transfer in mixed microbial communities. The fate of plasmids in microbial communities...... of microbial communities may be directly interconnected through transfer of BHR plasmids at a so far unrecognized level. The developed method furthermore enabled me to explore how agronomic practices may affect gene transfer in soil microbial communities. I compared bacterial communities extracted from plots...

Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri.

OpenAIRE

Vescovo, M; Morelli, L; Bottazzi, V

1982-01-01

Sixteen strains of Lactobacillus reuteri and 20 strains of Lactobacillus acidophilus were tested for resistance to 22 antibiotics by using commercially available sensitivity disks. Evidence suggesting linkage of these resistances to plasmids was obtained by "curing" experiments with acridine dyes and high growth temperatures. Examination of plasmid patterns of agarose gel electrophoresis provided further evidence of loss in plasmid DNA under curing conditions in some of the strains examined.

Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

Science.gov (United States)

Tran, Dinh Thi Minh; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Thi Kim; Nguyen, Tri Minh; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang Duc

2017-07-25

Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The β-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of

Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe.

Directory of Open Access Journals (Sweden)

Ayako Chino

Full Text Available BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC, is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp. No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different

46 CFR 221.85 - Hearing procedures.

Science.gov (United States)

2010-10-01

... § 221.85 Hearing procedures. (a) The Hearing Officer shall conduct a fair and impartial proceeding in... the authentication of any written exhibit or statement. (c) At the close of the Party's presentation...

MicroRNA-221 and -222 Regulate Radiation Sensitivity by Targeting the PTEN Pathway

International Nuclear Information System (INIS)

Zhang Chunzhi; Kang Chunsheng; Wang Ping; Cao Yongzhen; Lv Zhonghong; Yu Shizhu; Wang Guangxiu; Zhang Anling; Jia Zhifan; Han Lei; Yang Chunying; Ishiyama, Hiromichi; Teh, Bin S.; Xu Bo; Pu Peiyu

2011-01-01

Purpose: MicroRNAs (miRNAs) are noncoding RNAs inhibiting expression of numerous target genes by posttranscriptional regulation. miRNA-221 and miRNA-222 (miRNA-221/-222) expression is elevated in radioresistant tumor cell lines; however, it is not known whether and how miRNAs control cellular responses to ionizing irradiation. Methods and Materials: We used bioinformatic analyses, luciferase reporter assay, and genetic knockdown and biochemical assays to characterize the regulation pathways of miRNA-221/-222 in response to radiation treatment. Results: We identified the PTEN gene as a target of miRNA-221/-222. Furthermore, we found that knocking down miRNA-221/-222 by antisense oligonucleotides upregulated PTEN expression. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in tumor cells. Conclusions: miRNA-221/-222 control radiation sensitivity by regulating the PTEN/AKT pathway and can be explored as novel targets for radiosensitization.

Antibiotic resistance plasmids of Staphylococcus aureus and their clinical importance

International Nuclear Information System (INIS)

Lacey, R.W.

1975-01-01

A variety of plasmids were isolated physically, and most antibiotic resistance is thought to be plasmid mediated. A number of characters (e.g., resistance to erythromycin or methicillin, and production of pigment) are determined by genes that do not give clear indications of either plasmid or chromosomal location. Although the formation of a particular plasmid is probably, even in bacterial terms, a very rare event, once formed such an element can spread rapidly among the bacterial population. The spectacular increase in the incidence of penicillinase-producing hospital strains in the late 1940's could have been due in part to this process. Evidence is stronger, however, for the intercell transfer of recently isolated plasmids coding for resistance to fusidic acid (and penicillinase production), or for neomycin, or for tetracycline resistance. Study of bacterial plasmids can resolve fundamental biochemical problems, and give some insight into the life of the cell at the molecular level. But the immediate application of the study of staphylococcal plasmids may be directed towards improving the effectiveness of antibiotic therapy. The most important aspect of future anti-staphylococcal chemotherapy should thus be the limitation of the use of antibiotics, particularly for application to the skin and nose. (U.S.)

Comparison of arbitrarily primed-polymerase chain reaction and plasmid profiles typing of Pseudomonas aeruginosa strains from burn patients and hospital environment

International Nuclear Information System (INIS)

Japoni, A.; Farshad, S.; Alborzi, A.; Kalani, M.; Mohamadzadegan, R.

2007-01-01

Objective was to identify the strengths and weakness of arbitrary primed-polymerase chain reaction (A P-PCR) and plasmid profiles for typing of Pseudomonas aeruginosa (P. aeruginosa) and tracking of source of infections. Seventy-four strains of P. aeruginosa were isolated from burn patients and hospital environment between January to April 2003 in Ghotbadden Burn Hospital, Shiraz, Iran. The strains were classified by photo Capt Mw program, similarity and clustering of strains were assessed using NTSYS-PC version 2.02K software. Based on 50% and 64.7% and 67.5% similarity on the plotted dendrogram, 38 plasmid profiles were classified into: 2, 3 and 5 clusters, respectively. Photo Capt Mw program categorized AP-PCR products to 47 different types of 6 to 12 bands between 0.376 to 3.7 kb. Based on dendrogram pattern 3 levels (62 %, 81% and 84.6%) of similarity were selected. Using these criteria 2, 5and 11 clusters were obtained, respectively. As compared with plasmid profiles, AP-PCR analysis protocol is rapid, reproducible and differentiated the isolates with higher discrimination power. These results suggest that during admission of patients in burn center a limited number of common strains cross-contaminate burn victims. However, transmissions of infection from hospital environment to patients also occur in the minority of the victims. To control cross-contamination of the patient wounds with antibiotics resistant isolates, strong disinfection of patients' bathroom after scrubbing of each patient wounds is mandatory. (author)

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness.

Science.gov (United States)

Johnson, Timothy J; Singer, Randall S; Isaacson, Richard E; Danzeisen, Jessica L; Lang, Kevin; Kobluk, Kristi; Rivet, Bernadette; Borewicz, Klaudyna; Frye, Jonathan G; Englen, Mark; Anderson, Janet; Davies, Peter R

2015-05-15

IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

14 CFR 221.200 - Content and explanation of abbreviations, reference marks and symbols.

Science.gov (United States)

2010-01-01

..., reference marks and symbols. 221.200 Section 221.200 Aeronautics and Space OFFICE OF THE SECRETARY... § 221.200 Content and explanation of abbreviations, reference marks and symbols. (a) Content. The format..., reference marks and symbols. Abbreviations, reference marks and symbols which are used in the tariff shall...

Plasmid-Mediated Quinolone Resistance in Shigella flexneri Isolated From Macaques

Directory of Open Access Journals (Sweden)

Anthony J. Mannion

2018-03-01

Full Text Available Non-human primates (NHPs for biomedical research are commonly infected with Shigella spp. that can cause acute dysentery or chronic episodic diarrhea. These animals are often prophylactically and clinically treated with quinolone antibiotics to eradicate these possible infections. However, chromosomally- and plasmid-mediated antibiotic resistance has become an emerging concern for species in the family Enterobacteriaceae. In this study, five individual isolates of multi-drug resistant Shigella flexneri were isolated from the feces of three macaques. Antibiotic susceptibility testing confirmed resistance or decreased susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalosporins, gentamicin, tetracycline, ciprofloxacin, enrofloxacin, levofloxacin, and nalidixic acid. S. flexneri isolates were susceptible to trimethoprim-sulfamethoxazole, and this drug was used to eradicate infection in two of the macaques. Plasmid DNA from all isolates was positive for the plasmid-encoded quinolone resistance gene qnrS, but not qnrA and qnrB. Conjugation and transformation of plasmid DNA from several S. flexneri isolates into antibiotic-susceptible Escherichia coli strains conferred the recipients with resistance or decreased susceptibility to quinolones and beta-lactams. Genome sequencing of two representative S. flexneri isolates identified the qnrS gene on a plasmid-like contig. These contigs showed >99% homology to plasmid sequences previously characterized from quinolone-resistant Shigella flexneri 2a and Salmonella enterica strains. Other antibiotic resistance genes and virulence factor genes were also identified in chromosome and plasmid sequences in these genomes. The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes. To our knowledge, this is the first report of plasmid-mediated quinolone resistance in S. flexneri isolated from NHPs and warrants

DNA transformations of Candida tropicalis with replicating and integrative vectors.

Science.gov (United States)

Sanglard, D; Fiechter, A

1992-12-01

The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

Complete sequencing of IncI1 sequence type 2 plasmid pJIE512b indicates mobilization of blaCMY-2 from an IncA/C plasmid.

Science.gov (United States)

Tagg, Kaitlin A; Iredell, Jonathan R; Partridge, Sally R

2014-08-01

Sequencing of pJIE512b, a 92.3-kb IncI1 sequence type 2 (ST2) plasmid carrying bla(CMY-2), revealed a bla(CMY-2) context that appeared to have been mobilized from an IncA/C plasmid by the insertion sequence IS1294. A comparison with published plasmids suggests that bla(CMY-2) has been mobilized from IncA/C to IncI1 plasmids more than once by IS1294-like elements. Alignment of pJIE512b with the only other available IncI1 ST2 plasmid revealed differences across the backbones, indicating variability within this sequence type. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Novel assay to measure the plasmid mobilizing potential of mixed microbial communities

DEFF Research Database (Denmark)

Klümper, Uli; Droumpali, Ariadni; Dechesne, Arnaud

2014-01-01

Mobilizable plasmids lack necessary genes for complete conjugation and are therefore non-self-transmissible. Instead, they rely on the conjugation system of conjugal plasmids to be horizontally transferred to new recipients. While community permissiveness, the fraction of a mixed microbial...... community that can receive self-transmissible conjugal plasmids, has been studied, the intrinsic ability of a community to mobilize plasmids that lack conjugation systems is unexplored. Here, we present a novel framework and experimental method to estimate the mobilization potential of mixed communities. We...... of the donors receiving the conjugal plasmid in the first step. Further work is needed to establish how plasmid mobilization potential varies within and across microbial communities....

12 CFR 221.109 - Loan to open-end investment company.

Science.gov (United States)

2010-01-01

... CARRYING MARGIN STOCK (REGULATION U) Interpretations § 221.109 Loan to open-end investment company. In... customarily purchases stocks registered on a national securities exchange, the Board stated that in view of... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Loan to open-end investment company. 221.109...

Production and pharmaceutical formulation of plasmid DNA vaccines

NARCIS (Netherlands)

van der Heijden, I.

2013-01-01

Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human

Photoinduced silver nanoparticles/nanorings on plasmid DNA scaffolds.

Science.gov (United States)

Liu, Jianhua; Zhang, Xiaoliang; Yu, Mei; Li, Songmei; Zhang, Jindan

2012-01-23

Biological scaffolds are being actively explored for the synthesis of nanomaterials with novel structures and unexpected properties. Toroidal plasmid DNA separated from the Bacillus host is applied as a sacrificial mold for the synthesis of silver nanoparticles and nanorings. The photoirradiation method is applied to reduce Ag(I) on the plasmid. The nanoparticles are obtained by varying the concentration of the Ag(I) ion solution and the exposure time of the plasmid-Ag(I) complex under UV light at 254 nm and room temperature. It is found that the plasmid serves not only as a template but also as a reductant to drive the silver nucleation and deposition. The resulting nanoparticles have a face-centered cubic (fcc) crystal structure and 20-30 nm average diameter. The detailed mechanism is discussed, and other metals or alloys could also be synthesized with this method. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

Energy Technology Data Exchange (ETDEWEB)

Pollet, Rebecca M.; Ingle, James D.; Hymes, Jeff P.; Eakes, Thomas C.; Eto, Karina Yui; Kwong, Stephen M.; Ramsay, Joshua P.; Firth, Neville; Redinbo, Matthew R. (Curtin U.); (Sydney); (UNC)

2016-01-04

Antimicrobial resistance inStaphylococcus aureuspresents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at theoriTregion of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES proteinin vitro. Second, pSK156 and pCA347 are nonconjugativeStaphylococcus aureusplasmids that contain sequences similar to theoriTregion of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate theoriTsequences of these nonconjugative plasmidsin vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognateoriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variantoriTmimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-likeoriT. These data indicate that the conjugative relaxase intransmechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids.

IMPORTANCEUnderstanding the

Effect of vanillin on methylene blue plus light-induced single-strand breaks in plasmid pBR322 DNA.

Science.gov (United States)

Kumar, S S; Ghosh, A; Devasagayam, T P; Chauhan, P S

2000-09-20

The ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, in inhibiting photosensitization-induced single-strand breaks (ssbs) in plasmid pBR322 DNA has been examined in an in vitro system, independent of DNA repair/replication processes. Photosensitization of DNA with methylene blue, visible light and oxygen, induced ssbs resulting in the production of open circular form (OC form) in a concentration-dependent manner. The yield of OC form induced by photosensitization was increased several-fold by deuteration of the buffer and was found to be inhibited by sodium azide, a scavenger of singlet oxygen (1O(2)). Vanillin, per se, did not induce but inhibited photosensitization-induced ssbs in plasmid DNA, at millimolar concentrations. The inhibitory effect of vanillin was both concentration- and time-dependent. On a molar basis, vanillin was, however, less effective than trolox, a water-soluble analogue of alpha-tocopherol. Photosensitization by methylene blue system generates singlet oxygen, as one of the major components of ROS. Therefore, interaction of singlet oxygen with vanillin was investigated. The rate constant of vanillin with 1O(2) was estimated to be 5.93x10(7)M(-1)s(-1) and that of sodium azide as 2. 7x10(8)M(-1)s(-1). The present investigations show that vanillin can protect against photosensitization-induced ssbs in the plasmid pBR322 DNA, and this effect may partly be due to its ability to scavenge 1O(2).

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

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...

Resistant plasmid profile analysis of multidrug resistant Escherichia ...

African Journals Online (AJOL)

Multiple drug resistance isolates causing UTI has seri- ous implications for the empiric therapy against patho- genic isolates and for the possible co-selection of antimicrobial resistant mediated by multi drug resistant plasmids21,22. E. coli from clinical isolates are known to harbour plasmids of different molecular sizes23.

5 CFR 532.221 - Industries included in regular nonappropriated fund surveys.

Science.gov (United States)

2010-01-01

... nonappropriated fund surveys. 532.221 Section 532.221 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT... wholesalers. 44132 Tire dealers. 44311 Appliance, television, and other electronic stores. 44411 Home centers. 44611 Pharmacies and drug stores. 4471 Gasoline stations. 44814 Family clothing stores. 4521 Department...

Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure

Directory of Open Access Journals (Sweden)

Biryukova Irina V

2008-08-01

Full Text Available Abstract Background The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on in vitro and in vivo recombination processes have been developed. Results Here, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant E. coli strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-attB sites into desired points of the chromosome followed by two site-specific recombination processes: first, the φ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with φ80-attP-site, and second, the λ system is used for excision of inserted vector part, including the plasmid ori-replication and the marker, flanked by λ-attL/R-sites. Conclusion The developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry.

Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis.

Science.gov (United States)

Le Pihive, E; Blaha, D; Chenavas, S; Thibault, F; Vidal, D; Valade, E

2009-11-01

Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence.

Roles of three amino acids of capsid proteins in mink enteritis parvovirus replication.

Science.gov (United States)

Mao, Yaping; Su, Jun; Wang, Jigui; Zhang, Xiaomei; Hou, Qiang; Bian, Dawei; Liu, Weiquan

2016-08-15

Virulent mink enteritis parvovirus (MEV) strain MEV-LHV replicated to higher titers in feline F81 cells than attenuated strain MEV-L. Phylogenetic and sequence analyses of the VP2 gene of MEV-LHV, MEV-L and other strains in GenBank revealed two evolutionary branches separating virulent and attenuated strains. Three residues, 101, 232 and 411, differed between virulent and attenuated strains but were conserved within the two branches. Site-directed mutagenesis of the VP2 gene of infectious plasmids of attenuated strain MEV-L respectively replacing residues 101 Ile and 411 Ala with Thr and Glu of virulent strains (MEV-L I101T and MEV-L A411E) increased replication efficiency but still to lower levels than MEV-LHV. However, viruses with mutation of residue 232 (MEV-L I232V and MEV-L I101T/I232V/A411E) decreased viral transcription and replication levels. The three VP2 residues 101, 232 and 411, located on or near the capsid surface, played different roles in the infection processes of MEV. Copyright © 2016 Elsevier B.V. All rights reserved.

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

Science.gov (United States)

Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

2017-06-01

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

5 CFR 9901.221 - Classification requirements.

Science.gov (United States)

2010-01-01

... Section 9901.221 Administrative Personnel DEPARTMENT OF DEFENSE HUMAN RESOURCES MANAGEMENT AND LABOR RELATIONS SYSTEMS (DEPARTMENT OF DEFENSE-OFFICE OF PERSONNEL MANAGEMENT) DEPARTMENT OF DEFENSE NATIONAL..., qualifications, and other requirements of categories of jobs, and will make such descriptions and documentation...

Role of plasmids in Lactobacillus brevis BSO 464 hop tolerance and beer spoilage.

Science.gov (United States)

Bergsveinson, Jordyn; Baecker, Nina; Pittet, Vanessa; Ziola, Barry

2015-02-01

Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate.

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity.

Science.gov (United States)

Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno; Stensballe, Allan; Jensen, Ole N; Carter-Su, Christin

2004-06-01

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.

A Dual Role of P53 in Regulating Colistin-Induced Autophagy in PC-12 Cells

Directory of Open Access Journals (Sweden)

Ziyin Lu

2017-10-01

Full Text Available This study aimed to investigate the mechanism of p53 in regulating colistin-induced autophagy in PC-12 cells. Importantly, cells were treated with 125 μg/ml colistin for 12 and 24 h after transfection with p53 siRNA or recombinant plasmid. The hallmarks of autophagy and apoptosis were examined by real-time PCR and western blot, fluorescence/immunofluorescence microscopy, and electron microscopy. The results showed that silencing of p53 leads to down-regulation of Atg5 and beclin1 for 12 h while up-regulation at 24 h and up-regulation of p62 noted. The ratio of LC3-II/I and autophagic vacuoles were significantly increased at 24 h, but autophagy flux was blocked. The cleavage of caspase3 and PARP (poly ADP-ribose polymerase were enhanced, while PC-12-sip53 cells exposed to 3-MA showed down-regulation of apoptosis. By contrast, the expression of autophagy-related genes and protein reduced in p53 overexpressing cells following a time dependent manner. Meanwhile, there was an increase in the expression of activated caspase3 and PARP, condensed and fragmented nuclei were evident. Conclusively, the data supported that silencing of p53 promotes impaired autophagy, which acts as a pro-apoptotic induction factor in PC-12 cells treated with colistin for 24 h, and overexpression of p53 inhibits autophagy and accelerates apoptosis. Hence, it has been suggested that p53 could not act as a neuro-protective target in colistin-induced neurotoxicity.

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

Science.gov (United States)

Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

2017-01-01

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica

Directory of Open Access Journals (Sweden)

Maria Hoffmann

2017-08-01

Full Text Available Determinants of multidrug resistance (MDR are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI, and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about

ELIPGRID-PC: Upgraded version

International Nuclear Information System (INIS)

Davidson, J.R.

1995-12-01

Evaluating the need for and the effectiveness of remedial cleanup at waste sites often includes finding average contaminant concentrations and identifying pockets of contamination called hot spots. The standard tool for calculating the probability of detecting pockets of contamination called hot spots has been the ELIPGRID code of singer and Wickman. The ELIPGRID-PC program has recently made this algorithm available for an IBM reg-sign personal computer (PC) or compatible. A new version of ELIPGRID-PC, incorporating Monte Carlo test results and simple graphics, is herein described. Various examples of how to use the program for both single and multiple hot spot cases are given. The code for an American National Standards Institute C version of the ELIPGRID algorithm is provided, and limitations and further work are noted. This version of ELIPGRID-PC reliably meets the goal of moving Singer's ELIPGRID algorithm to the PC

Long- term manure exposure increases soil bacterial community potential for plasmid uptake

DEFF Research Database (Denmark)

Musovic, Sanin; Klümper, Uli; Dechesne, Arnaud

2014-01-01

Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive and main......Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive...... and maintain the plasmids. The community permissiveness increased up to 100% in communities derived from manured soil. While the plasmid transfer frequency was significantly influenced by both the type of plasmid and the agronomic treatment, the diversity of the transconjugal pools was purely plasmid dependent...

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

International Nuclear Information System (INIS)

Imura, Yoshiyuki; Molho, Melissa; Chuang, Chingkai; Nagy, Peter D.

2015-01-01

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

Energy Technology Data Exchange (ETDEWEB)

Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai; Nagy, Peter D., E-mail: pdnagy2@uky.edu

2015-10-15

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.

14 CFR 221.61 - Rules and regulations governing foreign air transportation.

Science.gov (United States)

2010-01-01

... governing foreign air transportation. Instead of being included in the fares tariffs, the rules and regulations governing foreign air transportation required to be filed by §§ 221.20 and 221.30 and/or... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Rules and regulations governing foreign air...

Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia

OpenAIRE

Yeow, Tee Cian; Wong, Won Fen; Sabet, Negar Shafiei; Sulaiman, Sofiah; Shahhosseini, Fatemeh; Tan, Grace Min Yi; Movahed, Elaheh; Looi, Chung Yeng; Shankar, Esaki M.; Gupta, Rishien; Arulanandam, Bernard P.; Hassan, Jamiyah; Abu Bakar, Sazaly

2016-01-01

Background The 7.5?kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis?infected patie...

Genome Stability of Lyme Disease Spirochetes: Comparative Genomics of Borrelia burgdorferi Plasmids

Energy Technology Data Exchange (ETDEWEB)

Casjens S. R.; Dunn J.; Mongodin, E. F.; Qiu, W.-G.; Luft, B. J.; Schutzer, S. E.; Gilcrease, E. B.; Huang, W. M.; Vujadinovic, M.; Aron, J. K.; Vargas, L. C.; Freeman, S.; Radune, D.; Weidman, J. F.; Dimitrov, G. I.; Khouri, H. M.; Sosa, J. E.; Halpin, R. A.; Fraser, C. M.

2012-03-14

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi {approx}900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short {le}20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

Plasmid Conjugation in E. coli and Drug Resistance | Igwe ...

African Journals Online (AJOL)

This study aimed at determining the antibiotics susceptibility pattern of E. coli isolates claimed to be multidrug resistance using disc diffusion method. It also determined the presence of transferable resistance plasmids through conjugation and evaluated the medical significance of plasmid encoding E. coli and drug ...

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements

Directory of Open Access Journals (Sweden)

Modesto Redrejo-Rodríguez

2017-11-01

Full Text Available Family B DNA polymerases (PolBs play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB, that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance.

周期型马来丝虫复合基因重组质粒和相应表达蛋白的免疫学研究%Immunology comparision of composite gene recombinant plasmid and expressed protein of periodical Brugia malayi

Institute of Scientific and Technical Information of China (English)

王慧; 方政; 徐倩; 陆施娟; 钱一言; 徐怿琳; 方浩; 徐邦生

2014-01-01

��的复合重组质粒/复合重组蛋白/CpG组小鼠淋巴细胞刺激增殖指数(1.629±0.235)高于复合重组蛋白组(1.248±0.110,P＜0.05);免疫4、6周后,复合重组质粒/复合重组蛋白/CpG组和复合重组质粒/CpG组小鼠血清IFN-γ水平[(101.660±5.101)、(178.265±7.139)mg/L,(102.067±3.722)、(115.148±6.031)mg/L]均高于复合重组蛋白组[(75.438±2.102)、(82.004±3.777) mg/L,P均＜0.05];免疫后6周,复合重组质粒/复合重组蛋白/CpG组和复合重组蛋白/CpG组的小鼠血清IL-4水平[(75.385±3.318)、(46.363±3.672)mg/L]均明显高于复合重组质粒/CpG组[(36.691±3.443)mg/L,P均＜0.05).结论 pcDNA3.1-BmCPI/BmGAPDH核酸疫苗和相应蛋白疫苗均可诱导BALB/c小鼠产生特异性体液和细胞免疫应答反应.核酸疫苗-蛋白疫苗联合免疫效果有明显的优势.%Objective To construct a plasmid DNA vector expressing cysteine protease inhibitor and glyceraldehydes-3-phosphate dehydrogenase of periodic Brugia malayi(BmCPI/BmGAPDH),and purify the recombinant protein after transfecting the vector into human cervical carcinoma cells(Hela) for expression.To make a comparison of immunity efficacy between the recombinant plasmid and the homologous protein and to a lay theoretic and experimental basis for developing novel anti-filarial genetic engineering vaccines.Methods The amplified genes BmCPI and BmGAPDH and a plasmid vector were double enzymes digested and ligated to construct a recombinant plasmid pcDNA3.1 (+)-BmCPI/BmGAPDH,and this plasmid was transfected to Hela cells after being identified.G418 was used for screening transfectants,and the monoclonal resistant cell strain was determined by RT-PCR and SDS-PAGE.The recombinant protein was purified by affnity chromatography and identified by Western blotting.Sixty BALB/c mice were divided into 5 groups,12 per group,and they were immunized at 2,4,and 6 weeks.Mice in control groups were injected with PBS 100 μ1 or pcDNA3.1 100 μg/CpG 30 μg,and mice in experimental groups were injected with pcDNA3.1-BmCPI/BmGAPDH 100 μg/CpG 30

PC-BLAS, PC Linear Algebra Subroutines

International Nuclear Information System (INIS)

Hanson, R.J.

1989-01-01

1 - Description of program or function: PC-BLAS is a highly optimized version of the Basic Linear Algebra Subprograms (BLAS), a standardized set of 38 routines that perform low-level operations on vectors of numbers in single- and double-precision real and complex arithmetic. Routines are included to find the index of the largest component of a vector, apply a Givens or modified Givens rotation, multiply a vector by a constant, determine the Euclidean length, perform a dot product, swap and copy vectors, and find the norm of a vector. 2 - Restrictions on the complexity of the problem: The number of components in any vector and the spacing or stride between their entries must not exceed 32,767 (2 15 -1). PC-BLAS will not work with an 80286 CPU operating in 'protected' mode

12 CFR 221.112 - Loans by bank in capacity as trustee.

Science.gov (United States)

2010-01-01

... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Loans by bank in capacity as trustee. 221.112... CARRYING MARGIN STOCK (REGULATION U) Interpretations § 221.112 Loans by bank in capacity as trustee. (a... been pledged as collateral for loans. Loans are not made, under the plan, from bank funds, and...

Alamethicin in lipid bilayers: combined use of X-ray scattering and MD simulations.

Science.gov (United States)

Pan, Jianjun; Tieleman, D Peter; Nagle, John F; Kucerka, Norbert; Tristram-Nagle, Stephanie

2009-06-01

We study fully hydrated bilayers of two di-monounsaturated phospholipids diC18:1PC (DOPC) and diC22:1PC with varying amounts of alamethicin (Alm). We combine the use of X-ray diffuse scattering and molecular dynamics simulations to determine the orientation of alamethicin in model lipids. Comparison of the experimental and simulated form factors shows that Alm helices are inserted transmembrane at high humidity and high concentrations, in agreement with earlier results. The X-ray scattering data and the MD simulations agree that membrane thickness changes very little up to 1/10 Alm/DOPC. In contrast, the X-ray data indicate that the thicker diC22:1PC membrane thins with added Alm, a total decrease in thickness of 4 A at 1/10 Alm/diC22:1PC. The different effect of Alm on the thickness changes of the two bilayers is consistent with Alm having a hydrophobic thickness close to the hydrophobic thickness of 27 A for DOPC; Alm is then mismatched with the 7 A thicker diC22:1PC bilayer. The X-ray data indicate that Alm decreases the bending modulus (K(C)) by a factor of approximately 2 in DOPC and a factor of approximately 10 in diC22:1PC membranes (P/L approximately 1/10). The van der Waals and fluctuational interactions between bilayers are also evaluated through determination of the anisotropic B compressibility modulus.

Peak Pc Prediction in Conjunction Analysis: Conjunction Assessment Risk Analysis. Pc Behavior Prediction Models

Science.gov (United States)

Vallejo, J.J.; Hejduk, M.D.; Stamey, J. D.

2015-01-01

Satellite conjunction risk typically evaluated through the probability of collision (Pc). Considers both conjunction geometry and uncertainties in both state estimates. Conjunction events initially discovered through Joint Space Operations Center (JSpOC) screenings, usually seven days before Time of Closest Approach (TCA). However, JSpOC continues to track objects and issue conjunction updates. Changes in state estimate and reduced propagation time cause Pc to change as event develops. These changes a combination of potentially predictable development and unpredictable changes in state estimate covariance. Operationally useful datum: the peak Pc. If it can reasonably be inferred that the peak Pc value has passed, then risk assessment can be conducted against this peak value. If this value is below remediation level, then event intensity can be relaxed. Can the peak Pc location be reasonably predicted?

Plasmids of Staphylococcus cohnii isolated from the intensive-care unit.

Science.gov (United States)

Szewczyk, E M; Rózalska, M; Cieślikowski, T; Nowak, T

2004-01-01

Numerous isolates of both subspecies of Staphylococcus cohnii were found in the environment of the intensive-care unit of a pediatric hospital. These isolates carried in their cells many plasmids, up to fourteen, of a wide range of sizes ( 56 kb). Striking was the occurrence of large plasmids not very common in staphylococci. These were present in > 80% of S. cohnii isolates. Fifty-two different plasmid profiles were found in 79 investigated isolates belonging to S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Isolates similar in plasmid profiles were grouped in antibiotic-resistance clusters established for 9 antibiotics (gentamicin, ciprofloxacin, clindamycin, erythromycin, tetracycline, chloramphenicol, mupirocin, trimethoprim-sulfamethoxazole, vancomycin) using the method of unweighted pair group mathematical averages (UPGMA). Many isolates were multiresistant to antibiotics and produced bacteriocins.

Sampling and Analysis Plan for the 221-U Facility

International Nuclear Information System (INIS)

Rugg, J.E.

1998-02-01

This sampling and analysis plan (SAP) presents the rationale and strategy for the sampling and analysis activities proposed to be conducted to support the evaluation of alternatives for the final disposition of the 221-U Facility. This SAP will describe general sample locations and the minimum number of samples required. It will also identify the specific contaminants of potential concern (COPCs) and the required analysis. This SAP does not define the exact sample locations and equipment to be used in the field due to the nature of unknowns associated with the 221-U Facility

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus

Directory of Open Access Journals (Sweden)

Michelle D. Rodriguez

2017-12-01

Full Text Available Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus. These three reporter plasmids are available through BEI Resources.

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus.

Science.gov (United States)

Rodriguez, Michelle D; Paul, Zubin; Wood, Charles E; Rice, Kelly C; Triplett, Eric W

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus . These three reporter plasmids are available through BEI Resources.

Quantitative comparison of HTLV-1 and HIV-1 cell-to-cell infection with new replication dependent vectors.

Directory of Open Access Journals (Sweden)

Dmitriy Mazurov

2010-02-01

Full Text Available We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.

Fast and efficient three-step target-specific curing of a virulence plasmid in Salmonella enterica.

Science.gov (United States)

de Moraes, Marcos H; Teplitski, Max

2015-12-01

Virulence plasmids borne by serovars of Salmonella enterica carry genes involved in its pathogenicity, as well as other functions. Characterization of phenotypes associated with virulence plasmids requires a system for efficiently curing strains of their virulence plasmids. Here, we developed a 3-step protocol for targeted curing of virulence plasmids. The protocol involves insertion of an I-SecI restriction site linked to an antibiotic resistance gene into the target plasmid using λ-Red mutagenesis, followed by the transformation with a temperature-sensitive auxiliary plasmid which carries I-SecI nuclease expressed from a tetracycline-inducible promoter. Finally, the auxiliary plasmid is removed by incubation at 42 °C and the plasmid-less strains are verified on antibiotic-containing media. This method is fast and very efficient: over 90 % of recovered colonies lacked their virulence plasmid.

Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

Science.gov (United States)

Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

2011-01-01

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

DEFF Research Database (Denmark)

Klümper, Uli; Riber, Leise; Dechesne, Arnaud

2014-01-01

and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse...

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

Science.gov (United States)

Pasion, S G; Brown, G W; Brown, L M; Ray, D S

1994-12-01

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

Knock-down of miR-221 and miR-222 in the radiosensitization of breast cancer cells

International Nuclear Information System (INIS)

Zhang Chunzhi; Kang Chunsheng; Cao Yongzhen; Pu Peiyu; Lu Zhonghong; Du Yue

2009-01-01

Objective: To investigate the radiosensitizing effect of knock-down of miR-221 miR-222 on MCF-7 human breast cancer cells and explore the possible mechanism. Methods: Antisense oligonucleotides of miR-221 and miR-222 (AS-miR-221 and AS-miR-222), mediated by lipofectamine, were transfected to MCF-7 cells to knock down miR-221 and miR-222, Northern blotting was conducted to detect the expression of miR-221 and miR-222 in transfected cells. The cell apoptosis was detected by flow cytometry and Caspase-3 and Caspase-7 activity assay. Clonogenic assay was used to measure the sensitizing enhancement ratio. Target genes of miR-221 and miR-222 relevant to radio-sensitivity were searched using bioinformatics analysis. The targeted protein expression was determined by Western blot analysis. Results: The expression of miR-221 and miR-222 in the AS-miR-221/222 cells determined by Northern blotting was significantly reduced. Compared with the control group, the cell apoptosis and mitotic cell death after the radiation were significantly higher in AS-miR-221/222 cells. The sensitizing enhancement ratio was 1.87. Based on bioinformatics analysis, PTEN was a target gene of miR-221 and miR-222 which could enhance the radiosensitivity of MCF-7 cells. In AS-miR-221/222 cells, the expression of PTEN was up-regulated while pAkt down-regulated. Conclusions: AS-miR-221 and AS-miR-222 may enhance the radiosensitivity of MCF-7 breast cancer cells by up-regulating the expression of PTEN. (authors)

Plasmids in Vibrio parahemolyticus strains isolated in Japan and Bangladesh with special reference to different distributions.

Science.gov (United States)

Arai, T; Ando, T; Kusakabe, A; Ullah, M A

1983-01-01

We surveyed plasmids in naturally occurring Vibrio parahemolyticus strains isolated in Japan and Bangladesh. Among the strains isolated in Japan, about half of the strains isolated from stools of patients of domestic diarrhea outbreaks as well as of travelers returning from East Asia were found to have plasmids, but no strains from foods had plasmids. In contrast, among the strains isolated in Bangladesh, none of the four strains isolated from patients had plasmids, but two out of eight strains isolated from water had plasmids, suggesting that plasmids are common in strains from the water in Bangladesh. All plasmids so far reported in V. parahemolyticus were detected in strains isolated from stools of patients. Incidences of plasmids in this organism were not so high in either area. In Japan, all plasmids were detected in strains from human intestines at 37 C, but in Bangladesh, where the temperature is around 30-40 C, the plasmids were detected in strains from the natural environment. These results suggested the possibility that these plasmids can come from different bacteria under rather high temperatures and that incidences of plasmids are influenced by the incidences of plasmids in bacteria present in the vicinity of V. parahemolyticus strains. None of these plasmids were found to have any relation to the biological characters tested.

Hepatitis B virus HBx protein localized to the nucleus restores HBx-deficient virus replication in HepG2 cells and in vivo in hydrodynamically-injected mice

International Nuclear Information System (INIS)

Keasler, Victor V.; Hodgson, Amanda J.; Madden, Charles R.; Slagle, Betty L.

2009-01-01

Identifying the requirements for the regulatory HBx protein in hepatitis B virus (HBV) replication is an important goal. A plasmid-based HBV replication assay was used to evaluate whether HBx subcellular localization influences its ability to promote virus replication, as measured by real time PCR quantitation of viral capsid-associated DNA. HBx targeted to the nucleus by a nuclear localization signal (NLS-HBx) was able to restore HBx-deficient HBV replication, while HBx containing a nuclear export signal (NES-HBx) was not. Both NLS-HBx and NES-HBx were expressed at similar levels (by immunoprecipitation and Western blotting), and proper localization of the signal sequence-tagged proteins was confirmed by deconvolution microscopy using HBx, NLS-HBx, and NES-HBx proteins fused to GFP. Importantly, these findings were confirmed in vivo by hydrodynamic injection into mice. Our results demonstrate that in these HBV replication assays, at least one function of HBx requires its localization to the nucleus.

Repairing and Upgrading Your PC

CERN Document Server

Thompson, Robert

2009-01-01

Repairing and Upgrading Your PC delivers start-to-finish instructions, simple enough for even the most inexperienced PC owner, for troubleshooting, repairing, and upgrading your computer. Written by hardware experts Robert Bruce Thompson and Barbara Fritchman Thompson, this book covers it all: how to troubleshoot a troublesome PC, how to identify which components make sense for an upgrade, and how to tear it all down and put it back together. This book shows how to repair and upgrade all of your PC's essential components.

Size unlimited markerless deletions by a transconjugative plasmid-system in Bacillus licheniformis.

Science.gov (United States)

Rachinger, Michael; Bauch, Melanie; Strittmatter, Axel; Bongaerts, Johannes; Evers, Stefan; Maurer, Karl-Heinz; Daniel, Rolf; Liebl, Wolfgang; Liesegang, Heiko; Ehrenreich, Armin

2013-09-20

Conjugative shuttle vectors of the pKVM series, based on an IncP transfer origin and the pMAD vector with a temperature sensitive replication were constructed to establish a markerless gene deletion protocol for Bacilli without natural competence such as the exoenzyme producer Bacillus licheniformis. The pKVM plasmids can be conjugated to strains of B. licheniformis and B. subtilis. For chromosomal gene deletion, regions flanking the target gene are fused and cloned in a pKVM vector prior to conjugative transfer from Escherichia coli to B. licheniformis. Appropriate markers on the vector backbone allow for the identification of the integration at the target locus and thereafter the vector excision, both events taking place via homologous recombination. The functionality of the deletion system was demonstrated with B. licheniformis by a markerless 939 bp in-frame deletion of the yqfD gene and the deletion of a 31 kbp genomic segment carrying a PBSX-like prophage. Copyright © 2013 Elsevier B.V. All rights reserved.

Sequential acquisition of R-plasmids in vivo by Salmonella typhimurium.

Science.gov (United States)

Platt, D J; Sommerville, J S; Gribben, J

1984-01-01

Salmonella typhimurium, resistant only to trimethoprim and sulphamethoxazole, was isolated from the faeces and blood of a chronic alcoholic patient in acute renal failure. The isolates harboured an 18 Md non-conjugative plasmid. He was dialysed peritoneally and treated with ampicillin; four days later there was no clinical improvement and his peritoneal dialysis fluid (PDF) had become infected. Salm. typhimurium was isolated from faeces and PDF. Both isolates were additionally resistant to ampicillin and contained two plasmids (55 Md and 18 Md). Therapy was changed to chloramphenicol and gentamicin was added to the PDF. Two weeks later Salm. typhimurium was again isolated from PDF and faeces. The PDF isolate was unchanged but 4% of the colonies isolated from this faecal specimen were resistant to chloramphenicol and had acquired an additional 62 Md plasmid. From all PDF and faecal specimens two different strains of Escherichia coli and one strain of Klebsiella pneumoniae were isolated which contained plasmids indistinguishable, on the basis of molecular weight and transferable resistance markers, from those acquired by Salm. typhimurium. The transferability of these plasmids in vitro to E. coli K12 and to the patient's initial Salm. typhimurium was studied and the results discussed.

50 CFR 221.34 - What is the law governing ex parte communications?

Science.gov (United States)

2010-10-01

... communications? 221.34 Section 221.34 Wildlife and Fisheries NATIONAL MARINE FISHERIES SERVICE, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE MARINE MAMMALS PRESCRIPTIONS IN FERC HYDROPOWER... communications? (a) Ex parte communications with the ALJ or his or her staff are prohibited in accordance with 5...

A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate.

Science.gov (United States)

Santos, Jefferson J S; Cordeiro, Marli T; Bertani, Giovani R; Marques, Ernesto T A; Gil, Laura H V G

2014-12-01

Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis.

The PC graphics handbook

CERN Document Server

Sanchez, Julio

2003-01-01

Part I - Graphics Fundamentals PC GRAPHICS OVERVIEW History and Evolution Short History of PC Video PS/2 Video Systems SuperVGA Graphics Coprocessors and Accelerators Graphics Applications State-of-the-Art in PC Graphics 3D Application Programming Interfaces POLYGONAL MODELING Vector and Raster Data Coordinate Systems Modeling with Polygons IMAGE TRANSFORMATIONS Matrix-based Representations Matrix Arithmetic 3D Transformations PROGRAMMING MATRIX TRANSFORMATIONS Numeric Data in Matrix Form Array Processing PROJECTIONS AND RENDERING Perspective The Rendering Pipeline LIGHTING AND SHADING Lightin

Non-additive costs and interactions alter the competitive dynamics of co-occurring ecologically distinct plasmids.

Science.gov (United States)

Morton, Elise R; Platt, Thomas G; Fuqua, Clay; Bever, James D

2014-03-22

Plasmids play an important role in shaping bacterial evolution and adaptation to heterogeneous environments. As modular genetic elements that are often conjugative, the selective pressures that act on plasmid-borne genes are distinct from those that act on the chromosome. Many bacteria are co-infected by multiple plasmids that impart niche-specific phenotypes. Thus, in addition to host-plasmid dynamics, interactions between co-infecting plasmids are likely to be important drivers of plasmid population dynamics, evolution and ecology. Agrobacterium tumefaciens is a facultative plant pathogen that commonly harbours two distinct megaplasmids. Virulence depends on the presence of the tumour-inducing (Ti) plasmid, with benefits that are primarily restricted to the disease environment. Here, we demonstrate that a second megaplasmid, the At plasmid, confers a competitive advantage in the rhizosphere. To assess the individual and interactive costs of these plasmids, we generated four isogenic derivatives: plasmidless, pAt only, pTi only and pAtpTi, and performed pairwise competitions under carbon-limiting conditions. These studies reveal a low cost to the virulence plasmid when outside of the disease environment, and a strikingly high cost to the At plasmid. In addition, the costs of pAt and pTi in the same host were significantly lower than predicted based on single plasmid costs, signifying the first demonstration of non-additivity between naturally occurring co-resident plasmids. Based on these empirically demonstrated costs and benefits, we developed a resource-consumer model to generate predictions about the frequencies of these genotypes in relevant environments, showing that non-additivity between co-residing plasmids allows for their stable coexistence across environments.

Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes

Science.gov (United States)

Lee, JunMo; Kim, Kyeong Mi; Yang, Eun Chan; Miller, Kathy Ann; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

2016-01-01

The integration of foreign DNA into algal and plant plastid genomes is a rare event, with only a few known examples of horizontal gene transfer (HGT). Plasmids, which are well-studied drivers of HGT in prokaryotes, have been reported previously in red algae (Rhodophyta). However, the distribution of these mobile DNA elements and their sites of integration into the plastid (ptDNA), mitochondrial (mtDNA), and nuclear genomes of Rhodophyta remain unknown. Here we reconstructed the complex evolutionary history of plasmid-derived DNAs in red algae. Comparative analysis of 21 rhodophyte ptDNAs, including new genome data for 5 species, turned up 22 plasmid-derived open reading frames (ORFs) that showed syntenic and copy number variation among species, but were conserved within different individuals in three lineages. Several plasmid-derived homologs were found not only in ptDNA but also in mtDNA and in the nuclear genome of green plants, stramenopiles, and rhizarians. Phylogenetic and plasmid-derived ORF analyses showed that the majority of plasmid DNAs originated within red algae, whereas others were derived from cyanobacteria, other bacteria, and viruses. Our results elucidate the evolution of plasmid DNAs in red algae and suggest that they spread as parasitic genetic elements. This hypothesis is consistent with their sporadic distribution within Rhodophyta. PMID:27030297

24 CFR 221.255 - Assignment option.

Science.gov (United States)

2010-04-01

... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Assignment option. 221.255 Section... Assignment option. (a) A mortgagee holding a mortgage insured pursuant to a conditional or firm commitment issued on or before November 30, 1983 has the option to assign, transfer and deliver to the Commissioner...

PcToll2 positively regulates the expression of antimicrobial peptides by promoting PcATF4 translocation into the nucleus.

Science.gov (United States)

Lan, Jiang-Feng; Zhao, Li-Juan; Wei, Shun; Wang, Yuan; Lin, Li; Li, Xin-Cang

2016-11-01

Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog PcATF4 from Procambarus clarkii, and analyzed the likely regulatory activity of PcATF4 in PcToll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. PcToll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, PcToll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus, PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the PcATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio, the translocation of PcATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio, the expression of ALF1 and ALF2 was also decreased while the expression of PcToll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of PcATF4, instead of the routine

30 CFR 250.221 - What environmental monitoring information must accompany the EP?

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 2 2010-07-01 2010-07-01 false What environmental monitoring information must accompany the EP? 250.221 Section 250.221 Mineral Resources MINERALS MANAGEMENT SERVICE, DEPARTMENT OF THE... necessary under the MMPA. (c) Flower Garden Banks National Marine Sanctuary (FGBNMS). If you propose to...

Natural plasmid transformation in a high-frequency-of transformation marine Vibrio strain

International Nuclear Information System (INIS)

Frischer, M.E.; Thurmond, J.M.; Paul, J.H.

1990-01-01

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 x 10 -9 and 3.4 x 10 -7 transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42,857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 x 10 -8 to 1.3 x 10 -4 transformants per recipient with plasmid DNA and at an average frequency of 8.3 x 10 -5 transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [ 3 H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations

Repair promoted by plasmid pKM101 is different from SOS repair

International Nuclear Information System (INIS)

Goze, A.; Devoret, R.

1979-01-01

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid PKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although Wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions. (Auth.)

33 CFR 135.221 - Reapplication for certification.

Science.gov (United States)

2010-07-01

... (CONTINUED) MARINE POLLUTION FINANCIAL RESPONSIBILITY AND COMPENSATION OFFSHORE OIL POLLUTION COMPENSATION FUND Financial Responsibility for Offshore Facilities § 135.221 Reapplication for certification. (a) If... certificate must be immediately submitted to the Fund Administrator in accordance with § 135.204. ...

12 CFR 221.124 - Purchase of debt securities to finance corporate takeovers.

Science.gov (United States)

2010-01-01

... securities to finance corporate takeovers. (a) Petitions have been filed with the Board raising questions as... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Purchase of debt securities to finance corporate takeovers. 221.124 Section 221.124 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF...

Phase I feasibility study for the Canyon disposition initiative (221-U facility)

International Nuclear Information System (INIS)

1997-08-01

The objective of the Phase I Feasibility Study (FS) presented in this document is to provide decision makers sufficient information on the remedial alternatives specific to the disposition of the 221-U Canyon Building (221-U Facility) located at the Hanford Site, and to determine which alternatives are viable for further detailed analysis

CoPc and CoPcF16 on gold: Site-specific charge-transfer processes

Directory of Open Access Journals (Sweden)

Fotini Petraki

2014-04-01

Full Text Available Interface properties of cobalt(II phthalocyanine (CoPc and cobalt(II hexadecafluoro-phthalocyanine (CoPcF16 to gold are investigated by photo-excited electron spectroscopies (X-ray photoemission spectroscopy (XPS, ultraviolet photoemission spectroscopy (UPS and X-ray excited Auger electron spectroscopy (XAES. It is shown that a bidirectional charge transfer determines the interface energetics for CoPc and CoPcF16 on Au. Combined XPS and XAES measurements allow for the separation of chemical shifts based on different local charges at the considered atom caused by polarization effects. This facilitates a detailed discussion of energetic shifts of core level spectra. The data allow the discussion of site-specific charge-transfer processes.

Comparative metagenomic analysis of plasmid encoded functions in the human gut microbiome

Directory of Open Access Journals (Sweden)

Marchesi Julian R

2010-01-01

Full Text Available Abstract Background Little is known regarding the pool of mobile genetic elements associated with the human gut microbiome. In this study we employed the culture independent TRACA system to isolate novel plasmids from the human gut microbiota, and a comparative metagenomic analysis to investigate the distribution and relative abundance of functions encoded by these plasmids in the human gut microbiome. Results Novel plasmids were acquired from the human gut microbiome, and homologous nucleotide sequences with high identity (>90% to two plasmids (pTRACA10 and pTRACA22 were identified in the multiple human gut microbiomes analysed here. However, no homologous nucleotide sequences to these plasmids were identified in the murine gut or environmental metagenomes. Functions encoded by the plasmids pTRACA10 and pTRACA22 were found to be more prevalent in the human gut microbiome when compared to microbial communities from other environments. Among the most prevalent functions identified was a putative RelBE toxin-antitoxin (TA addiction module, and subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes. A broad phylogenetic distribution of RelE toxin genes was observed in gut associated bacterial species (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, but no RelE homologues were identified in gut associated archaeal species. We also provide indirect evidence for the horizontal transfer of these genes between bacterial species belonging to disparate phylogenetic divisions, namely Gram negative Proteobacteria and Gram positive species from the Firmicutes division. Conclusions The application of a culture independent system to capture novel plasmids from the human gut mobile metagenome, coupled with subsequent comparative metagenomic analysis, highlighted the unexpected prevalence of plasmid encoded functions in the gut microbial ecosystem. In

12 CFR 221.122 - Applicability of margin requirements to credit in connection with Insurance Premium Funding...

Science.gov (United States)

2010-01-01

... in connection with Insurance Premium Funding Programs. 221.122 Section 221.122 Banks and Banking...) Interpretations § 221.122 Applicability of margin requirements to credit in connection with Insurance Premium... with insurance premium funding programs. The inquiries are included in a set of guidelines in the...

Plant-inducible virulence promoter of the Agrobacterium tumefaciens Ti plasmid

NARCIS (Netherlands)

Okker, Robert J.H.; Spaink, Herman; Hille, Jacques; Brussel, Ton A.N. van; Lugtenberg, Ben; Schilperoort, Rob A.

1984-01-01

Agrobacterium tumefaciens is the causative agent of crown gall, a plant tumour that can arise on most species of dicotyledonous plants. The tumour-inducing capacity of the bacterium requires the presence of a large plasmid, designated the Ti plasmid, which itself contains two regions essential for

Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

Science.gov (United States)

Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

2016-04-01

Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

Directory of Open Access Journals (Sweden)

Yanping Wen

Full Text Available Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4% were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2 and 32 isolates (17.0% were positive for aac(6'-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6'-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05. In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05. All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6'-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.

Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar.

Directory of Open Access Journals (Sweden)

Yo Sugawara

Full Text Available The bacterial enzyme New Delhi metallo-β-lactamase hydrolyzes almost all β-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-β-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1, IncX3 plasmids harboring blaNDM-4 (n = 2 or blaNDM-7 (n = 1, IncFII plasmids harboring blaNDM-4 (n = 1 or blaNDM-5 (n = 3, and a multireplicon F plasmid harboring blaNDM-5 (n = 1. Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.

PC/104 Embedded IOCs at Jefferson Lab

International Nuclear Information System (INIS)

Yan, Jianxun; Allison, Trent; Witherspoon, Sue; Cuffe, Anthony

2009-01-01

Jefferson Lab has developed embedded IOCs based on PC/104 single board computers (SBC) for low level control systems. The PC/104 IOCs run EPICS on top of the RTEMS operating system. Two types of control system configurations are used in different applications, PC/104 SBC with commercial PC/104 I/O cards and PC/104 SBC with custom designed FPGA-based boards. RTEMS was built with CEXP shell to run on the PC/104 SBC. CEXP shell provides the function of dynamic object loading, which is similar to the widely used VxWorks operating system. Standard software configurations were setup for PC/104 IOC application development to provide a familiar format for new projects as well as ease the conversion of applications from VME based IOCs to PC/104 IOCs. Many new projects at Jefferson Lab are going to employ PC/104 SBCs as IOCs and some applications have already been running them for accelerator operations. The PC/104 - RTEMS IOC provides a free open source Real-Time Operating System (RTOS), low cost/maintenance, easily installed/ configured, flexible, and reliable solution for accelerator control and 12GeV Upgrade projects.

Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

Directory of Open Access Journals (Sweden)

Kayla A Boortz

Full Text Available Elevated fasting blood glucose (FBG has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln, rs149663725 (Gly114Arg and rs2232326 (Ser324Pro SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu, rs138726309 (His177Tyr, rs2232323 (Tyr207Ser rs374055555 (Arg293Trp, rs2232326 (Ser324Pro, rs137857125 (Pro313Leu and rs2232327 (Pro340Leu SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich 167RRRSQSPRR175 Domain Is Critical for HBV Replication

Science.gov (United States)

Kim, Taeyeung; Shin, Bo-Hye; Park, Gil-Soon; Park, Sun; Chwae, Yong-Joon; Shin, Ho-Joon; Kim, Kyongmin

2012-01-01

To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221–262 amino acids of DHBV C protein, in place of 146–185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221–241 and 251–262 amino acids of DHBV C, in place of HBV C 146–166 and 176–185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242–250 of DHBV C (242RAGSPLPRS 250) introduced in place of 167–175 of HBV C (167RRRSQSPRR 175) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich 167RRRSQSPRR175 domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important. PMID:22911745

Radiation environmental monitoring and assessment of plant-221 site ten years after decommissioning

International Nuclear Information System (INIS)

Li Yang; Gu Zhijie; Pan Wei; Ren Xiaona; Hu Xiaolin; She Haiqiang

2011-01-01

More than 10 years have passed since nuclear facility decommissioning practice for Plant-221 finished. Environmental radiation monitoring and post assessment of the decommissioning site of Plant-221 was carried out during 2003-2006, which was organized by Department of Environmental Protection and executed by China Institute for Radiation Protection, Environmental Radiation Monitoring station of Qinghai Province, etc. It shows that the decommissioning practice for Plant-221 complied with relevant limits for decommissioning, and its environmental radiation situation has not had significant change in general after 10 years, and the potential impact to the public and the environmental is acceptable. (authors)

Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum

Energy Technology Data Exchange (ETDEWEB)

Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian [Ohio State Univ., Columbus, OH (United States). William G. Lowrie Dept. of Chemical and Biomolecular Engineering; Tang, I-Ching [Bioprocessing Innovative Company, Dublin, OH (United States)

2012-01-15

Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at {proportional_to}6.0. (orig.)

Insights into dynamics of mobile genetic elements in hyperthermophilic environments from five new Thermococcus plasmids.

Directory of Open Access Journals (Sweden)

Mart Krupovic

Full Text Available Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1, with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

PC'eren på arbejde. 3. udgave

DEFF Research Database (Denmark)

Andersen, Povl Erik Rostgård

En generel og ikke-programspecifik introduktion til edb-området med speciel fokus på pc'er og pc-programmel.......En generel og ikke-programspecifik introduktion til edb-området med speciel fokus på pc'er og pc-programmel....

Two novel conjugative plasmids from a single strain of Sulfolobus

NARCIS (Netherlands)

Erauso, G.; Stedman, K.M.; Werken, van de H.J.G.; Zillig, W.; Oost, van der J.

2006-01-01

Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4, The plasmids were separated from each other and transferred into Sulfolobus soltataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number

Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

NARCIS (Netherlands)

Diepeningen, van A.D.; Debets, A.J.M.; Slakhorst-Wandel, S.M.; Hoekstra, R.F.

2008-01-01

Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with

Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

NARCIS (Netherlands)

van Diepeningen, Anne D; Debets, Alfons J M; Slakhorst, S Marijke; Hoekstra, Rolf F

Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with

Enhancing access to alcohol use disorder pharmacotherapy and treatment in primary care settings: ADaPT-PC.

Science.gov (United States)

Hagedorn, Hildi J; Brown, Randall; Dawes, Michael; Dieperink, Eric; Myrick, Donald Hugh; Oliva, Elizabeth M; Wagner, Todd H; Wisdom, Jennifer P; Harris, Alex H S

2016-05-10

rates of AUD pharmacotherapy prescribing and rates of referral to PCMHI and SUD specialty care), and (4) interpretive FE (post-implementation interviews with champions and PC providers). Analysis of FE data will be guided by the Consolidated Framework for Implementation Research (CFIR). If demonstrated to be successful, this implementation strategy will provide a replicable, feasible, and relative low-cost method for integrating AUD treatment services into PC settings, thereby increasing access to AUD treatment.

Targeted delivery of chemically modified anti-miR-221 to hepatocellular carcinoma with negatively charged liposomes

Directory of Open Access Journals (Sweden)

Zhang W

2015-07-01

Full Text Available Wendian Zhang,1 Fangqi Peng,1 Taotao Zhou,1 Yifei Huang,2 Li Zhang,3 Peng Ye,4 Miao Lu,1 Guang Yang,5 Yongkang Gai,1 Tan Yang,1 Xiang Ma,1 Guangya Xiang1 1School of Pharmacy, Tongji Medical College, 2Department of Pharmacy, 3Department of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 4Department of Pharmacy, Wuhan University, Renmin Hospital, 5School of Medicine, Jianghan University, Wuhan, People’s Republic of China Abstract: Hepatocellular carcinoma (HCC is one of the leading causes of cancer-related death. Gene therapy was established as a new strategy for treating HCC. To explore the potential delivery system to support the gene therapy of HCC, negatively charged liposomal delivery system was used to deliver miR-221 antisense oligonucleotide (anti-miR-221 to the transferrin (Tf receptor over expressed HepG2 cells. The liposome exhibited a mean particle size of 122.5 nm, zeta potential of -15.74 mV, anti-miR-221 encapsulation efficiency of 70%, and excellent colloidal stability at 4°C. Anti-miR-221-encapsulated Tf-targeted liposome demonstrated a 15-fold higher delivery efficiency compared to nontargeted liposome in HepG2 cells in vitro. Anti-miR-221 Tf-targeted liposome effectively delivered anti-miR-221 to HepG2 cells, upregulated miR-221 target genes PTEN, P27kip1, and TIMP3, and exhibited greater silencing efficiency over nontargeted anti-miR-221 liposome. After intravenous injection into HepG2 tumor-bearing xenografted mice with Cy3-labeled anti-miR-221 Tf-targeted liposome, Cy3-anti-miR-221 was successfully delivered to the tumor site and increased the expressions of PTEN, P27kip1, and TIMP3. Our results demonstrate that the Tf-targeted negatively charged liposome could be a potential therapeutic modality in the gene therapy of human HCC. Keywords: transferrin, gene, HCC, target delivery system, anionic liposome 

Plasmid profiles and antibiotic susceptibility patterns of Staphylococcus aureus isolates from Nigeria.

Science.gov (United States)

Olukoya, D K; Asielue, J O; Olasupo, N A; Ikea, J K

1995-06-01

In an investigation into the problems of infections due to Staphylococcus aureus in Nigeria, 100 strains were isolated from various hospitals in Lagos. The strains were screened for the presence of plasmids and for susceptibility to antimicrobial agents. Plasmids were extracted by modification of the method of Takahashi and Nagono[1]. The plasmids were diverse in nature. The strains were found to be highly resistant to commonly prescribed antibiotics.

Giant linear plasmids in Streptomyces: a treasure trove of antibiotic biosynthetic clusters.

Science.gov (United States)

Kinashi, Haruyasu

2011-01-01

Many giant linear plasmids have been isolated from Streptomyces by using pulsed-field gel electrophoresis and some of them were found to carry an antibiotic biosynthetic cluster(s); SCP1 carries biosynthetic genes for methylenomycin, pSLA2-L for lankacidin and lankamycin, and pKSL for lasalocid and echinomycin. Accumulated data suggest that giant linear plasmids have played critical roles in genome evolution and horizontal transfer of secondary metabolism. In this review, I summarize typical examples of giant linear plasmids whose involvement in antibiotic production has been studied in some detail, emphasizing their finding processes and interaction with the host chromosomes. A hypothesis on horizontal transfer of secondary metabolism involving giant linear plasmids is proposed at the end.

Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4.

Science.gov (United States)

Cao, Qing-Hua; Shao, Huan-Huan; Qiu, Hui; Li, Tao; Zhang, Yi-Zheng; Tan, Xue-Mei

2017-03-01

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.

Transfer of conjugative plasmids among bacteria under environmentally relevant conditions

DEFF Research Database (Denmark)

Musovic, Sanin

Mobile genetiske elementer (f.eks. plasmider), der ofte bærer ekstra funktioner såsom antibiotikaresistens, eller kataboliske- og xenobiotiske nedbrydnings gener, antages at have en meget vigtigt evolutionær rolle for bakterier. I denne PhD afhandling undersøgte jeg størrelsen af plasmid overførs...

Isolation and properties of plasmids from Deinococcus radiodurans Sark

International Nuclear Information System (INIS)

Sjarief, S.H.; Kikuchi, Masahiro; Kurita, Hiromi; Kitayama, Shigeru; Watanabe, Hiroshi.

1990-05-01

Radioresistant bacterium, Deinococcus radiodurans, can repair completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 kGy. In order to reveal the repair mechanism, it is necessary to develop a cloning vector available for the genetic analysis. We tried to isolate plasmids from D.radiodurans Sark strain. In the present paper the isolation and properties of plasmids were described. (author)

IBM PC/IX operating system evaluation plan

Science.gov (United States)

Dominick, Wayne D. (Editor); Granier, Martin; Hall, Philip P.; Triantafyllopoulos, Spiros

1984-01-01

An evaluation plan for the IBM PC/IX Operating System designed for IBM PC/XT computers is discussed. The evaluation plan covers the areas of performance measurement and evaluation, software facilities available, man-machine interface considerations, networking, and the suitability of PC/IX as a development environment within the University of Southwestern Louisiana NASA PC Research and Development project. In order to compare and evaluate the PC/IX system, comparisons with other available UNIX-based systems are also included.

Analysis of plasmid profiling as a method for rapid differentiation of food-associated Clostridium perfringens strains.

Science.gov (United States)

Jones, M K; Iwanejko, L A; Longden, M S

1989-09-01

Plasmid analysis of over 120 strains of Clostridium perfringens, isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6.2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.

Presence of Glycopeptide-Encoding Plasmids in Enterococcal Isolates from Food and Humans in Denmark

DEFF Research Database (Denmark)

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

2011-01-01

developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat...... and animals in Europe and is rarely found in E. faecalis. This article describes the first characterization of MGE from vanA mediated E. faecalis, thus linking this resistance genotype to pheromone responding plasmids....

50 CFR 221.47 - What are the requirements for subpoenas and witness fees?

Science.gov (United States)

2010-10-01

... and witness fees? 221.47 Section 221.47 Wildlife and Fisheries NATIONAL MARINE FISHERIES SERVICE... requirements for subpoenas and witness fees? (a) Request for subpoena. (1) Except as provided in paragraph (a... return it to the party on whose behalf the subpoena was served. (c) Witness fees. (1) A party who...

Infectious alphavirus production from a simple plasmid transfection+

Directory of Open Access Journals (Sweden)

Olson Ken E

2011-07-01

Full Text Available Abstract We have developed a new method for producing infectious double subgenomic alphaviruses from plasmids transfected into mammalian cells. A double subgenomic Sindbis virus (TE3'2J was transcribed from a cytomegalovirus PolII promoter, which results in the production of infectious virus. Transfection of as little as 125 ng of plasmid is able to produce 1 × 108 plaque forming units/ml (PFU/ml of infectious virus 48 hours post-transfection. This system represents a more efficient method for producing recombinant Sindbis viruses.

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

OpenAIRE

Shrago, A W; Chassy, B M; Dobrogosz, W J

1986-01-01

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance.

Science.gov (United States)

Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

2003-07-05

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable.

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance

International Nuclear Information System (INIS)

Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

2003-01-01

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable

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

Junior PC-Kørekort

DEFF Research Database (Denmark)

Hansbøl, Mikala; Mathiasen, Helle

udviklingsprojekt med forsøg med DANSK IT's Junior PC-kørekort® som metode og redskab til at evaluere og dokumentere elevernes IT-kompetencer. Projektet blev gennemført i perioden fra 2002-2003 og afsluttet i foråret 2003. Rapporten er baseret på kvalitative case studier af elever og læreres arbejde med Junior PC......-kørekort® prøverne. DANSK IT's Junior PC-kørekort® kan betragtes som et forsøg på at etablere en bestemt fælleskulturel tilgang til elevernes IT-kompetencer i folkeskolen. Ud fra et situeret læringsperspektiv problematiserer rapporten anvendelsen af de standardiserede prøver som metode til at dokumentere og evaluere...... elevernes IT-kompetencer. Rapporten (2MB) fylder 80 sider ekskl. bilag. Bemærk, at forskningsrapporten handler om Dansk IT's Junior PC-kørekort - ikke at forveksle med det IT-bevis for folkeskolens elever, som Undevisningsministeriet har udviklet. Dette IT-bevis koncept skiftede navn i januar 2004, hvor...

Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

DEFF Research Database (Denmark)

Porse, Andreas; Schønning, Kristian; Munck, Christian

2016-01-01

sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions...... consistently followed by all evolved E. coli lineages exposes a trade-off between horizontal and vertical transmission that may ultimately limit the dissemination potential of clinical multidrug resistance plasmids in these hosts....

Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Ikai, K.; Tano, K.; Ohnishi, T.; Nozu, K.

1985-01-01

The repair of UV-irradiated DNA of plasmid YEp13 was studied in the incision defective strains by measurement of cell transformation frequency. In Saccharomyces cerevisiae, rad1,2,3 and 4 mutants could repair UV-damaged plasmid DNA. In Escherichia coli, uvrA mutant was unable to repair UV-damaged plasmid DNA; however, pretreatment of the plasmid with Micrococcus luteus endonuclease increased repair. It was concluded that all the mutations of yeast were probably limited only to the nuclear DNA. (author)

Phosphatidylcholine (PC) biosynthesis in pancreatic islets of Langerhans

International Nuclear Information System (INIS)

Hoffman, J.M.; Laychock, S.G.

1986-01-01

Islets of Langerhans isolated from rat pancreata were incubated with [ 14 C]choline to determine the biosynthesis of PC by the CDP choline to determine the biosynthesis of PC by the CDPcholine pathway. Recovery of [ 14 C]PC in islet membranes was time-related, and stimulated by glucose (17mM) during 60 min. The rate of PC synthesis was constant during 60 min with glucose stimulation. In contrast, the sulfonylurea tolbutamide (2 mM) reduced the recovery of [ 14 C]choline in PC, and 8-bromo-cyclic AMP (5 mM) did not significantly affect [ 14 C]PC recovery. Incubation of islets in Ca 2+ -free medium enhanced glucose-stimulated recovery of [ 14 C]choline-labeled PC due to the inhibition of phospholipase and phospholipid hydrolysis. Inhibition of CTP:phosphocholine cytidylyltransferase with 5-deoxy-5'-isobutylthioadenosine (SIBA) reduced [ 14 C]PC levels and insulin release in a concentration dependent manner. Treatment with SIBA also reduced Mg 2+ -dependent Ca 2+ -ATPase activity in islet microsomes. Quantitation of membrane PC showed that glucose stimulation did not alter islet P levels. Thus, islet PC biosynthesis is linked to glucose stimulation and contributes to the maintenance of PC levels in membranes undergoing exocytosis and phospholipid hydrolysis. Adequate PC levels support Ca 2+ pump activity and secretory mechanisms

Plasmid DNA Analysis of Pasteurella multocida Serotype B isolated from Haemorrhagic Septicaemia outbreaks in Malaysia

Directory of Open Access Journals (Sweden)

Jamal, H.

2005-01-01

Full Text Available A total of 150 purified isolates of Pasteurella multocida serotype B were used (Salmah, 2004 for plasmid DNA curing experiment to determine hyaluronidase activity, antibiotic resistance pattern (ARP and mice lethality test (LD50 for their role of pathogenicity. A plasmid curing experiment was carried out by using the intercalating agent; ethidium bromide and rifampicin, where it was found all the plasmids had been cured (plasmidless from Pasteurella multocida. All of these plasmidless isolates maintained their phenotypic characteristics. They showed the same antibiotic resistancepattern as before curing, produced hyaluronidase and possessed lethality activity in mice when injected intraperitoneally(i.p. Based on this observation, the antibiotic resistance, hyaluronidase activity and mice virulence could probably be chromosomal-mediated. Plasmids were detected 100% in all P. multocida isolates with identical profile of 2 plasmids size 3.0 and 5.5 kb. No large plasmids could be detected in all isolates. Since all the isolates appeared to have identicalplasmid profiles, they were subjected to restriction enzyme(RE analysis. From RE analysis results obtained, it can be concluded that the plasmid DNA in serotype B isolates are identical. Only 4 of 32 REs were found to cleave these plasmids with identical restriction fingerprints; BglII, HaeIII, RsaI and SspI. From RE analysis results, it can be concluded that the plasmid DNA isolates are identical. This plasmid might not played any role in pathogenicity of Pasteurella multocida serotype B, however this information is important for the construction of shuttle vectors in genetic studies of the pathogenicity of haemorrhagic septicaemia(HS.

Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA

Science.gov (United States)

Hershfield, Vickers; Boyer, Herbert W.; Yanofsky, Charles; Lovett, Michael A.; Helinski, Donald R.

1974-01-01

DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage ϕ80pt190 (trp+) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp- strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the ϕ80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes. Images PMID:4610576

Centromere pairing by a plasmid-encoded type I ParB protein

DEFF Research Database (Denmark)

Ringgaard, Simon; Löwe, Jan; Gerdes, Kenn

2007-01-01

The par2 locus of Escherichia coli plasmid pB171 encodes two trans-acting proteins, ParA and ParB, and two cis-acting sites, parC1 and parC2, to which ParB binds cooperatively. ParA is related to MinD and oscillates in helical structures and thereby positions ParB/parC-carrying plasmids regularly......, hence identifying the N terminus of ParB as a requirement for ParB-mediated centromere pairing. These observations suggest that centromere pairing is an important intermediate step in plasmid partitioning mediated by the common type I loci....

Quorum-Dependent Mannopine-Inducible Conjugative Transfer of an Agrobacterium Opine-Catabolic Plasmid

Science.gov (United States)

Wetzel, Margaret E.; Kim, Kun-Soo; Miller, Marilyn; Olsen, Gary J.

2014-01-01

The Ti plasmid in Agrobacterium tumefaciens strain 15955 carries two alleles of traR that regulate conjugative transfer. The first is a functional allele, called traR, that is transcriptionally induced by the opine octopine. The second, trlR, is a nonfunctional, dominant-negative mutant located in an operon that is inducible by the opine mannopine (MOP). Based on these findings, we predicted that there exist wild-type agrobacterial strains harboring plasmids in which MOP induces a functional traR and, hence, conjugation. We analyzed 11 MOP-utilizing field isolates and found five where MOP induced transfer of the MOP-catabolic element and increased production of the acyl-homoserine lactone (acyl-HSL) quormone. The transmissible elements in these five strains represent a set of highly related plasmids. Sequence analysis of one such plasmid, pAoF64/95, revealed that the 176-kb element is not a Ti plasmid but carries genes for catabolism of MOP, mannopinic acid (MOA), agropinic acid (AGA), and the agrocinopines. The plasmid additionally carries all of the genes required for conjugative transfer, including the regulatory genes traR, traI, and traM. The traR gene, however, is not located in the MOP catabolism region. The gene, instead, is monocistronic and located within the tra-trb-rep gene cluster. A traR mutant failed to transfer the plasmid and produced little to no quormone even when grown with MOP, indicating that TraRpAoF64/95 is the activator of the tra regulon. A traM mutant was constitutive for transfer and acyl-HSL production, indicating that the anti-activator function of TraM is conserved. PMID:24363349

Plasmids which make their host bacteria mutable as well as resistant to ultraviolet irradiation