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PubMed2005

Ultrasound enhances in vivo tumor expression of plasmid DNA by PEG-introduced cationized dextran.

This study is an investigation to experimentally confirm whether or not ultrasound (US) irradiation is effective in enhancing the in vivo gene expression of plasmid DNA in tumor. Dextran was cationized by introducing spermine to the hydroxyl groups to allow to polyionically complex with a plasmid DNA. The cationized dextran prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have an active ester and methoxy groups at each terminal, to obtain cationized dextran with different percentages of PEG introduced. Various cationized dextrans with or without PEG introduction were mixed with a plasmid DNA of LacZ to form cationized dextran-plasmid DNA complexes. Electrophoretical examination revealed that the plasmid DNA was complexed both with the cationized dextran and PEG-introduced cationized dextran, irrespective of the PEG introduction percentage, although the higher N/P ratio was needed for plasmid DNA complexation with the latter. By complexation with the cationized dextran, the zeta potential of plasmid DNA was changed to be positive. The charge of PEG-introduced cationized dextran-plasmid DNA complexes became close to 0 mV as their percentage of PEG introduced increased, although the molecular size was about 250 nm, irrespective of the PEG introduction. When cationized dextran-plasmid DNA complexes with or without PEG introduction were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass and the subsequent US irradiation to the tumor mass percutaneously, the PEG-introduced cationized dextran-plasmid DNA complex plus US irradiation enhanced the tumor level of gene expression to a significantly high extent compared with the cationized dextran-plasmid DNA complex and free plasmid DNA with or without US irradiation. The enhanced level depended on the time period and timing of US irradiation. Fluorescent microscopic studies revealed that the localization of plasmid DNA and the gene expression were observed in the tumor tissue injected with the PEG-introduced cationized dextran-plasmid DNA complex plus the subsequent US irradiation. We conclude that complexation with the PEG-introduced cationized dextran combined with US irradiation is a promising way to target the plasmid DNA to the tumor for gene expression.

Animals↗

PubMed2006

Evolution of the virulence plasmids of non-typhoid Salmonella and its association with antimicrobial resistance.

Among more than 2,500 serovars, eight contain a virulence plasmid, including medically important Salmonella enterica serovars Choleraesuis, Dublin, Enteritidis, and Typhimurium. These serovar-specific virulence plasmids vary in size, but all contain the spv operon, which plays a role in the expression of the virulence. Genetically, these virulence plasmids are likely derived from a common ancestral plasmid possessing virulence-related genes and loci. Based on the analysis of the available DNA sequences of the plasmids, the phylogenetic path may be split into two: pSPV (virulence plasmid of S. Gallinarum-Pullorum) acquires an incompatibility-related locus that differs from that of the others. At some point, pSCV (virulence plasmid of S. Choleraesuis) and pSDV (virulence plasmid of S. Dublin) lose oriT by recombination or simply by deletion, making the two unable to be mobilized. On the other hand, pSEV (virulence plasmid of S. Enteritidis) also loses some DNA by deletion but not as extensively as pSCV, and therefore pSEV is closest to pSTV (virulence plasmid of S. Typhimurium) both genetically and biologically. The pSTV shows the least alternation during the evolution. There are two types of pSDV. pSDVu recombines with non-virulence 36.6-kb plasmid to acquire additional incompatibility trait to form pSDVr. Recent reports indicated that S. Choleraesuis and S. Typhimurium could generate different types of hybrid plasmids, which consisted of the serovar-specific virulence plasmid and an array of resistance gene cassettes. The recombination gives Salmonella a survival advantage in an unfavorable drug environment. The integration of resistance genes and additional replicons into a Salmonella virulence plasmid constitutes a new and interesting example of plasmid evolution and poses a serious threat to public health.

Animals↗

PubMed2005

Replication regions of Sinorhizobium meliloti plasmids.

The replication (rep) regions of small plasmids from three Sinorhizobium meliloti strains were cloned by marker rescue. Two unique replication regions were identified, one of which was common to two different strains. Plasmid pBB83 carried a 7.2 kbp rep region from a 42 kbp plasmid, and pBB84 carried a 4.5 kbp rep region from a 36 kbp plasmid. The cloned rep regions were of different compatibility types, and were capable of displacing their parent plasmids from S. meliloti. Neither could function in a PolA- strain of Escherichia coli. The cloned replication regions were less stable in S. meliloti than their parent plasmids. The rep genes for each plasmid were localized to less than 2.5 kbp segments. Sequencing data revealed that the pBB83 Rep protein is uncommon, with partial identity to a protein encoded by a plasmid from S. meliloti GR4 [Mercado-Blanco, J., Olivares, J., 1994. The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids. Plasmid 32, 75-79]. However, the cloned DNA fragment also contains a truncated segment of the common repABC genes, suggesting that the parent plasmid contained two sets of replication genes. Other genes and an IS-element within the insert are most closely related to sequences derived from the Rhizobiaceae family, suggesting that the plasmid has a limited host range. In contrast, the pBB84 rep region contained genes similar to those associated with several broad host-range plasmids, and its Rep protein is related to that of a Pseudomonas aeruginosa broad host-range plasmid, pVS1 [Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Walsh, U., O'Gara, F., Haas, D., 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol. Plant-Microbe Interact. 13, 232-237]. The pBB84 rep region also includes a probable origin of replication, consisting of DNA boxes flanking a series of direct repeats and an AT-rich sequence.

Blotting, Southern↗

PubMed2001

Poly(lactic acid)-poly(ethylene glycol) nanoparticles as new carriers for the delivery of plasmid DNA.

The purpose of the present work was to produce and characterize poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles (size lower than 300 nm) containing a high loading of plasmid DNA in a free form or co-encapsulated with either poly(vinyl alcohol) (PVA) or poly(vinylpyrrolidone) (PVP). The plasmid alone or with PVA or PVP was encapsulated by two different techniques: an optimized w/o/w emulsion-solvent evaporation technique as well as by a new w/o emulsion-solvent diffusion technique. Particle size, zeta potential, plasmid DNA loading and in vitro release were determined for the three plasmid-loaded formulations. The influence of the initial plasmid loadings (5, 10, 20 microg plasmid DNA/mg PLA-PEG) on those parameters was also investigated. The plasmid loaded into the nanoparticles and released in vitro was quantified by fluorimetry and the different molecular forms were identified by gel electrophoresis. PLA-PEG nanoparticles containing plasmid DNA in a free form or co-encapsulated with PVA or PVP were obtained in the range size of 150-300 nm and with a negative zeta potential, both parameters being affected by the preparation technique. Encapsulation efficiencies were high irrespective of the presence of PVA or PVP (60-90%) and were slightly affected by the preparation technique and by the initial loading. The final plasmid DNA loading in the nanoparticles was up to 10-12 microg plasmid DNA/mg polymer. Plasmid DNA release kinetics varied depending on the plasmid incorporation technique: nanoparticles prepared by the w/o diffusion technique released their content rapidly whereas those obtained by the w/o/w showed an initial burst followed by a slow release for at least 28 days. No significant influence of the plasmid DNA loading and of the co-encapsulation of PVP or PVA on the in vitro release rate was observed. In all cases the conversion of the supercoiled form to the open circular and linear forms was detected. In conclusion, plasmid DNA can be very efficiently encapsulated, either in a free form or in combination with PVP and PVA, into PLA-PEG nanoparticles. Additionally, depending on the processing conditions, these nanoparticles release plasmid DNA either very rapidly or in a controlled manner.

Chemical Phenomena↗

PubMed2006

Inducible Escherichia coli fermentation for increased plasmid DNA production.

Bacterial plasmids are the vectors of choice for DNA vaccines and short-term gene therapeutics. Growing plasmid DNA by microbial (Escherichia coli) fermentation is usually combined with alkaline lysis/chromatography methods of purification. To date, typical plasmid fermentation media and processes result in yields of 100-250 mg of plasmid DNA/l of culture medium, using standard high-copy pUC origin-containing plasmids. In order to address this initial and yield-limiting upstream step, we identified novel fermentation control parameters for fed-batch fermentation. The resulting fermentation strategies significantly increased specific plasmid yield with respect to cell mass while enhancing plasmid integrity and maintaining supercoiled DNA content. Fed-batch fermentation yield exceeding 1000 mg of plasmid DNA/l was obtained after reduction of plasmid-mediated metabolic burden during growth, and yields up to 1500 mg of plasmid DNA/l have been achieved with optimized plasmid backbones. Interestingly, by inducing high plasmid levels after sufficient biomass accumulation at low temperature and restricted growth, cells were able to tolerate significantly higher plasmid quantities than cells grown by conventional processes. This 5-10-fold increase in plasmid yield dramatically decreases plasmid manufacturing costs and improves the effectiveness of downstream purification by reducing the fraction of impurities.

Bioreactors↗

PubMed1983

Mitotic and meiotic stability of linear plasmids in yeast.

Circular recombinant DNA plasmids that contain autonomously replicating sequences (ARSs) are maintained in extrachromosomal form in transformed yeast cells. However, these plasmids are unstable, being rapidly lost from cells growing without selection. Although the stability of such a plasmid can be increased by the presence of yeast centromere DNA (CEN), even CEN plasmids are lost at a high rate compared to a bona fide yeast chromosome. Natural yeast chromosomes are linear molecules; therefore, we have asked if linearization can improve the stability of recombinant DNA plasmids. Linear plasmids with and without yeast CENs were constructed in vitro by using termini from the extrachromosomal ribosomal DNA (rDNA) of the ciliated protozoan Tetrahymena thermophila as "telomeres." These linear plasmids transformed yeast at high frequency and were maintained as linear extrachromosomal molecules during mitotic growth. Moreover, linear plasmids containing CENs were also transmitted through meiosis: these plasmids segregate predominantly 2+:2- at the first meiotic division, indicating that Tetrahymena rDNA termini can provide telomere function during yeast meiosis. Linear plasmids without CENs were about as stable in mitosis as the comparable circular plasmid. Thus, the Tetrahymena rDNA termini have no marked positive or negative effect on the mitotic stability of ARS plasmids. However, linear plasmids containing CENs are three to four times less stable in mitotic cells than circular CEN plasmids. This decrease in stability is not due to a functional change in the centromere itself; rather, linearization of a CEN plasmid has a direct detrimental effect on its mitotic stability. These results may reflect the existence of spatial constraints on the positions of centromeres and telomeres, constraints which must be satisfied to achieve stable segregation of chromosomes during mitosis.

Animals↗

PubMed1988

Transfer of the Ti plasmid from Agrobacterium tumefaciens into Escherichia coli cells.

We have screened strains of Agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline Ti plasmid pTiC58 during conjugation. The Ti plasmid derivatives obtained could be transferred not only to A. tumefaciens but also to E. coli cells. The Ti plasmid cannot survive as a freely replicating plasmid in E. coli, but it can occasionally integrate into the E. coli chromosome. However, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) into the T-DNA provides an indicator for all transfer events into E. coli cells, providing fd gene 2 protein is present in these cells. This viral protein causes the excision of one copy of the pfd plasmid and allows its propagation in the host cell. By using this specially designed Ti plasmid, which was also made constitutive in transfer functions, we found plasmid exchange among A. tumefaciens strains and between A. tumefaciens and E. coli cells to be equally efficient. A Ti plasmid with repressed transfer functions was transferred to E. coli with a rate similar to the low frequency at which it was transferred to A. tumefaciens. The expression of transfer functions of plasmid RP4 either in A. tumefaciens or in E. coli did not increase the transfer of the Ti plasmid into E. coli cells, nor did the addition of acetosyringone, an inducer of T-DNA transfer to plant cells. The results show that A. tumefaciens can transfer the Ti plasmid to E. coli with the same efficiency as within its own species. Conjugational transmission of extrachromosomal DNA like the narrow-host-range Ti plasmid may often not only occur among partners allowing propagation of the plasmid, but also on a 'try-all' basis including hosts which do not replicate the transferred DNA.

Chromosome Mapping↗

PubMed1993

Molecular comparison of the IncX plasmids allows division into IncX1 and IncX2 subgroups.

We have investigated molecular relationships and evolution of plasmids classified genetically to incompatibility (Inc) group X, in particular by comparison of plasmids from the pre-antibiotic era (PAE) with contemporary R-plasmids. On the basis of restriction analysis, R6K, the best-described and 'prototype' plasmid of the IncX group, exhibited little similarity with the other plasmids in this Inc group. Other contemporary IncX R-plasmids exhibited a substantial degree of interrelationship, and were also related to PAE IncX plasmids. When the origin of plasmid replication of R6K was used as a replicon probe, R6K was the only plasmid tested which exhibited homology. Other contemporary and PAE IncX plasmids exhibited homology with the origin of plasmid R485. These data suggest that the IncX group should be subdivided. R485 may be regarded as representative of the major subgroup present before and after the advent of antibiotic selection pressure. Plasmids of this subgroup, IncX1, possess an internal region which yields five characteristic EcoRV fragments. R6K may be regarded as representative of subgroup IncX2, of which it is presently the sole well-described member. The antibiotic resistances encoded by contemporary IncX R-plasmids are due to insertion of identifiable transposons in progenitor plasmids identical to the R485 subgroup of PAE IncX plasmids.

Biological Evolution↗

PubMed1986

Studies of host-plasmid interactions in recombinant microorganisms.

Plasmid genes redirect some components of cellular metabolism into synthesis of plasmid gene products and additional plasmids. The stoichiometric and kinetic implications of these host-plasmid interactions have been investigated theoretically and experimentally. Using known pathway energetics, maximum theoretical yield factors based on ATP, glucose, and O2 have been estimated for recombinant Escherichia coli and compared with corresponding estimates for host cells alone, indicating major changes in carbon and energetic stoichiometry in recombinant cells in cases of high cloned gene expression. The influence of the number of plasmids in recombinant E. coli has been experimentally characterized using a series of pMB1 derivatives stably propagated at copy numbers from 12 to 408. Recombinant cell growth rate declines monotonically as plasmid content increases as does efficiency of plasmid gene expression. A detailed metabolically structured single-cell model for E. coli has successfully simulated these trends. Interrelationships among number of plasmids per cell, induction of expression of a plasmid gene, and recombinant population growth rate have been experimentally delineated for Saccharomyces cerevisiae containing plasmid pLGSD5 and derivatives in which the 2-micron origin of replication has been replaced by a cloned ARS1 sequence or its deletion fragments. The CEN4 centromere sequence has been included in some of these plasmids to provide more regular segregation. Specific growth rate of these recombinant yeasts exhibits a maximum as a function of plasmid content, an effect attributed to the interplay between beneficial effects of the plasmid in selective medium and parasitic effects on metabolism at larger plasmid content or with more plasmid gene expression activity. The yeast strains investigated exhibit substantial segregational instability that was characterized using a rapid-flow cytometry measurement based upon single-cell deletion of E. coli beta-galactosidase activity in recombinant cells.

DNA, Recombinant↗

PubMed1982

Genetic and molecular studies of plasmids coding for colonization factor antigen I and heat-stable enterotoxin in several escherichia coli serotypes.

Plasmids coding for colonization factor antigen I (CFA/I) and heat-stable enterotoxin (ST) were identified in 10 strains of human enterotoxigenic Escherichia coli. The strains, which belonged to serogroups O63, O114, O128, and O153, were isolated in Bangladesh, Latin America, Spain, and South Africa. Two strains produced heat-labile enterotoxin in addition to ST. CFA/I-ST plasmids were mobilized from two O128 strains into E. coli K-12 with the R factor R1-19K-. Like the prototype CFA/I-ST plasmid NTP113, mobilized previously from an E. coli O78 strain into K-12, these two plasmids were non-autotransferring. All 10 CFA/I-ST plasmids were incompatible with NTP113 and had molecular weights ranging from 59 X 10(6) to 72 X 10(6). The molecular properties of seven of these plasmids were compared with those of six CFA/I-ST plasmids previously mobilized from O78 strains from Ethiopia, South Africa, and Bangladesh and with those of one plasmid coding for CFA/I, ST and heat-labile enterotoxin from a South African strain of serogroup O63. Digestion with the restriction endonuclease HindIII showed that several plasmids had very similar fragment patterns and two were identical. Generally, a larger proportion of HindIII fragments were of common size in digests of plasmids identified in strains from related geographical areas, regardless of serogroup. However, all except one plasmid shared five or six HindIII fragments of the same size, one of which had been shown previously to be involved in CFA/I production. There was at least 90% DNA homology between CFA/I-ST plasmids with a molecular weight of about 58 X 10(6) from O78 strains from different sources. Most of the DNA sequences of these plasmids were present in a larger CFA/I-ST plasmid (72 X 10(6) from an O128 strain. The results of genetic and molecular studies suggest that CFA/I and ST production is determined by very similar plasmids in different serogroups of human enterotoxigenic E. coli from several sources.

Antigens, Bacterial↗

PubMed1975

RP1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids.

Incompatibility group P plasmids demonstrate strong entry exclusion properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range.

Biological Evolution↗

PubMed1979

Transformation-derived Neisseria gonorrhoeae plasmids with altered structure and function.

Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.

Conjugation, Genetic↗

PubMed1980

Relationship of plasmids responsible for hairy root and crown gall tumorigenicity.

Three strains of Agrobacterium rhizogenes were examined for plasmids. Strains 15834 and A4 contained essentially identical large plasmids, pAr15834c and pArA4c, respectively (approximately 260 x 10(6) daltons). These plasmids can dissociate to two smaller plasmid species. Strain TR105 contained only a single plasmid, which was homologous with the dissociation product of pAr15834c, pAr15834b. Plasmid pAr15834c shared little overall sequence homology with other Ti plasmids. One region of conserved homology between pAr15834c and a region of the octopine type plasmid pTiB6806 which contains oncogenicity functions was detected. Lower levels of homology were detected with sequences which are distributed throughout 65% of pTiB6806. Homology with the so-called common deoxyribonucleic acid in the integrated plasmid deoxyribonucleic acid region was detected only after lowering the stringency of hybridization (Tm, -41 degrees C). Furthermore, the A. rhizogenes plasmid is compatible with other Ti plasmids. Therefore, the results suggest that the virulence plasmids of A. rhizogenes are functionally similar to other Ti plasmids, yet have diverged sufficiently from an ancestral Ti plasmid that they now represent a distinct plasmid type based on homology, compatibility, and virulence.

Base Sequence↗

PubMed1982

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction.

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.

DNA, Recombinant↗

PubMed1983

Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology. Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes. The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1. pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells. Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids. The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis.

Base Sequence↗

PubMed1998

[Plasmid profile typing of Salmonella typhimurium and its distribution in some provinces and cities of China].

OBJECTIVE: To study the relationship between the infection and prevalence of Salmonella typhimurium. METHODS: From 1980 to 1995, when Salmonella typhimurium infection was prevalent in north and south China, a total of 2,655 isolates of Salmonella typhimurium were collected from 19 provinces, municipalities, and autonomous regions and Hong Kong Special Administrative Region for plasmid profile analysis. The profiles consisting of some small plamids were recorded with capital letters A to J, and the profile which was free from small plasmid was recorded with capital letter O. Large plasmid in molecular weights of 140-20 Mdal showing in profiles was recorded with Arabic numerals 9 to 0 respectively. Having two or more than two large plasmids, the profile was recorded with two or more Arabic numerals. RESULTS: The 2,655 strains were divided into 11 plasmid profile groups and 168 plasmid profile types. Of 52 plasmid profile types frequently isolated, plasmid profile type O 4 was distributed extensively in 16 provinces, municipalities, and autonomous rehions and Hong Kong. Plasmid profile type O 5 was isolated in 5 provinces and Hong Kong, mostly in Jiangxi and Hubei. Plasmid profile type J 4 was isolated in 7 provinces, municipalities and autonomous regions, mostly in Jiangxi. Five plasmid profile types of Group A caused a big prevalence of nosocomial infection in Henan. Type A 6 was also isolated in Xinjiang and Anhui. Six plasmid profile types of Group E were prevalent in Xinjiang predominantely, three types caused nosocomial infection in Shanghai, type E 82 caused nosocomial infection and food poisoning in Liaoning. Type F 4 was isolated in 4 provinces, mostly in jiangxi and Fujian. Many plasmid profile types were prevalent local. Types B 5, BCEG 60, BE 6, B' 0 G 6, G 5 and EG 6 were prevalent in Jiangxi, Types IF'4, IF'H40 and F 9864 were prevalent in Sandong, Type IF'4 was also isolated in Jiangsu and Hubei, Type IF 40 in Anhui, Type BH 6 in Huibei, and Type EH 5 in Fujian. CONCLUSION: Plasmid profile analysis showed the same types in the prevance but different plasmid profiles in different provinces.

China↗

PubMed2003

Study of recombinant micro-organism populations characterized by their plasmid content per cell using a segregated model.

Numerous observations from recombinant systems have shown that properties such as the specific cell growth rate and the plasmid-free cell formation rate are related, not only to the average plasmid content per cell, but also to the plasmid distribution within a population. The plasmid distribution in recombinant cultures can have an effect on the culture productivity that cannot be modelled using average values of the overall culture. The prediction of the behaviour of a plasmid content distribution and its causes and effects can only be studied using segregated models. A segregated model that describes populations of recombinant cells characterized by their plasmid content distribution has been developed. This model includes critical causes of recombinant culture instability such as the plasmid partition mechanism at cell division, plasmid replication kinetics and the effect of the plasmid content on the specific growth rate. The segregated model allows investigation of the effect of each of these causes and that of the plasmid content distribution on the observable behaviour of a recombinant culture. The effect of two partitioning mechanisms (Gaussian distribution and binomial distribution) on culture stability was investigated. The Gaussian distribution is slightly more stable. A small plasmid replication rate constant results in a very unstable culture even after short periods of time. This instability is dramatically improved for a larger value of this constant, hence improving protein synthesis. For a very narrow initial plasmid distribution, a given plasmid replication rate and partitioning mechanism can become broad even after a relatively short period of time. In contrast, a very "broad" initial distribution gave rise to a "Gamma-like" distribution profile. If we compare the results obtained in the simulations of the segregated model with those of the non-segregated one (average model), the latter model predicts much more stable behaviour, thus these average models cannot predict culture instability with the same precision. When compared with the experimental results, the segregated model was able to predict the practical behaviour with accuracy even in a system with a high plasmid content per cell and a high rate of plasmid-free cell formation which could not be achieved with a non-segregated model.

Journal Article↗

PubMed1985

Worldwide distribution of the conjugative Clostridium perfringens tetracycline resistance plasmid, pCW3.

The aim of this study was to test the hypothesis that all conjugative R-plasmids of Clostridium perfringens are closely related to the previously characterized tetracycline resistance plasmid, pCW3. Fourteen conjugative R-plasmids derived from 11 C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analyzed. Eleven of the plasmids encoded tetracycline resistance while three carried both tetracycline and chloramphenicol resistance. Each of these plasmids was compared, by restriction analysis, to the reference plasmid, pCW3. Seven of the tetracycline resistance plasmids had EcoRI, XbaI, and ClaI restriction profiles that were identical to those of the corresponding pCW3 digests. The seven remaining R-plasmids were different from pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contained at least 17 kb of DNA that also was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The three tetracycline and chloramphenicol resistance plasmids frequently lost a 6-kb chloramphenicol resistance segment during conjugation. Cloning experiments showed that the chloramphenicol resistance determinant was expressed in Escherichia coli and that the chloramphenicol resistance gene of one of these plasmids, pIP401, was contained within a 1.5-kb region of the 6-kb deletion segment. Hybridization analysis indicated that the deletion segment of pIP401 was related to those of the other two chloramphenicol resistance plasmids. During the course of this study, conjugative R-plasmids which appear to be identical to pCW3 or closely related to pCW3 were identified from C. perfringens strains from human, animal and environmental sources in five countries. It is concluded that C. perfringens strains in humans and animals throughout the world have overlapping gene pools and that all the conjugative C. perfringens R-plasmids examined probably evolved from a pCW3-like element.

Animal Diseases↗