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PubMed2006

Enhanced brain targeting efficiency of intranasally administered plasmid DNA: an alternative route for brain gene therapy.

Recently, nasal administration has been studied as a noninvasive route for delivery of plasmid DNA encoding therapeutic or antigenic genes. Here, we examined the brain targeting efficiency and transport pathways of intranasally administered plasmid DNA. Quantitative polymerase chain reaction (PCR) measurements of plasmid DNA in blood and brain tissues revealed that intranasally administered pCMVbeta (7.2 kb) and pN2/CMVbeta (14.1 kb) showed systemic absorption and brain distribution. Following intranasal administration, the beta-galactosidase protein encoded by these plasmids was significantly expressed in brain tissues. Kinetic studies showed that intranasally administered plasmid DNA reached the brain with a 2,595-fold higher efficiency than intravenously administered plasmid DNA did, 10 min post-dose. Over 1 h post-dose, the brain targeting efficiencies were consistently higher for intranasally administered plasmid DNA than for intravenously administered DNA. To examine how plasmid DNA enters the brain and moves to the various regions, we examined tissues from nine brain regions, at 5 and 10 min after intranasal or intravenous administration of plasmid DNA. Intravenously administered plasmid DNA displayed similar levels of plasmid DNA in the nine different regions, whereas, intranasally administered plasmid DNA exhibited different levels of distribution among the regions, with the highest plasmid DNA levels in the olfactory bulb. Moreover, plasmid DNA was mainly detected in the endothelial cells, but not in glial cells. Our results suggest that intranasally applied plasmid DNA may reach the brain through a direct route, possibly via the olfactory bulb, and that the nasal route might be an alternative method for efficiently delivering plasmid DNA to the brain.

Administration, Intranasal↗
PubMed1998

The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina.

A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids.

Ascomycota↗
PubMed1976

Characterization of a replication mutant of the bacteriocinogenic plasmid Clo DF13.

In a previous paper (Kool, A.J. and Nijkamp, H.J.J. (1974) J. Bacteriol. 120, 569--578) the isolation of a mutant of the bacteriocinogenic plasmid Clo DF13-Rep3, has been described. It was observed that cells harbouring the wild type plasmid synthesize more plasmid DNA cells harbouring the wild type plasmid. This paper deals with the characterization of the nature of this plasmid-specific mutation. The following properties of the Clo DF13-Rep3 mutant plasmid could be observed: 1. The plasmid-specific mutation did not lead to a significant change in the sedimentation value of Clo DF13 DNA. 2. The specific rate of Clo DF13-Rep3 DNA synthesis (expressed as the number of plasmid DNA molecules synthesized per min) is on average seven times the specific rate of wild type Clo DF13 DNA synthesis. 3. Also chromosomeless minicells, harbouring the Clo DF 13-Rep 3 plasmid, contain about seven times more plasmid DNA as wild type Clo DF13 harbouring minicells. 4. The replication time of the Clo DF13-Rep3 plasmid is approx. 90 s at 30 degrees C and does not differ significantly from the replication time of the wild type plasmid. 5. The Rep3 mutation did not alter the dependence of Clo DF13 plasmid replication on the dnaA and dnaC gene products. 6. The plasmid-specific mutation is cis-dominant over wild type. The data presented in this paper indicate that this mutant plasmid is not affected in the elongation but in the initiation of plasmid DNA replication.

Cell Division↗
PubMed1976

Mutants of the killer plasmid of Saccharomyces cerevisiae dependent on chromosomal diploidy for expression and maintenance.

Mutants of the killer plasmid of Saccharomyces cerevisiae have been isolated that depend upon chromosomal diploidy for the expression of plasmid functions and for replication or maintenance of the plasmid itself. These mutants are not defective in any chromosomal gene needed for expression or replication of the killer plasmid.--Haploids carrying these mutant plasmids (called d for diploid-depen;ent) are either unable to kill or unable to resist being killed or both and show frequent loss of the plasmid. The wild-type phenotype (K+R+) is restored by mating the d plasmid-carrying strain with either (a) a wild-type sensitive strain which apparently has no killer plasmid; (b) a strain which has been cured of the killer plasmid by growth at elevated temperature; (c) a strain which has been cured of the plasmid by growth in the presence of cycloheximide; (d) a strain which has lost the plasmid because it carries a mutation in a chromosomal mak gene; or (e) a strain of the opposite mating type which carries the same d plasmid and has the same defective phenotype, indicating that the restoration of the normal phenotype is not due to recombination between plasmid genomes or complementation of plasmid or chromosomal genes.--Sporulation of the phenotypically K+R+ diploids formed in matings between d and wild-type nonkiller strains yields tetrads, all four of whose haploid spores are defective for killing or resistance or maintenance of the plasmid or a combination of these. Every defective phenotype may be found among the segregants of a single diploid clone carrying a d plasmid. These defective segregants resume the normal killer phenotype in the diploids formed when a second round of mating is performed, and the segregants from a second round of meiosis and sporulation are again defective.

Crosses, Genetic↗
PubMed1997

Plasmid analysis of Neisseria gonorrhoeae isolates and dissemination of tetM genes in southern Africa 1993-1995.

One group (145 isolates) of Neisseria gonorrhoeae was collected from municipal clinics in Bloemfontein in 1994 and a second group (65 isolates) in 1995. Penicillin and tetracycline MICs were determined and plasmid analysis performed to monitor antimicrobial susceptibilities in conjunction with the occurrence of plasmids in these isolates. The prevalence of penicillin resistance caused by beta-lactamase plasmids remained constant at 9% during the study period. Three high-level tetracycline-resistant strains (MICs 16 mg/L), the first to be detected in South Africa, were isolated in 1994. Although there was a reduction in the percentage of isolates harbouring 24.5 MDa conjugative plasmids (from 79% in 1994 to 46% in 1995), this was partially counteracted by an increase in TetM-encoding conjugative plasmids (25.2 MDa) from 2% to 18.5%. The tetM genes of 13 isolates shown to exhibit high-level tetracycline resistance were characterized as the American type. The American-type tetracycline resistance plasmid was demonstrated in 11 isolates. Digestion with Bg/l showed that two isolates harboured tetM-encoding plasmids that differed from the American- and Dutch-type plasmids described previously: one isolate contained a plasmid that produced two fragments of different sizes from those of the American-type plasmid and the second isolate possessed an American/Dutch hybrid plasmid. Auxotyping/serotyping and random amplified polymorphic DNA analysis revealed a predominant tetracycline-resistant family (NR/IA-6, genomic group I) in Bloemfontein. As there is a high incidence of chlamydial infections in southern Africa requiring tetracycline therapy, selective pressures exist in the environment for the maintenance and rapid spread of high-level tetracycline-resistant N. gonorrhoeae. It is possible that tetM genes may have emanated from Botswana and/or Namibia to Bloemfontein. The establishment of high-level tetracycline-resistant N. gonorrhoeae in Bloemfontein was seen to be complex as a related group of strains was identified, plasmid dissemination was evident and two new TetM-encoding plasmids were demonstrated. The appearance of these TetM-encoding plasmids indicates either that the American- and Dutch-type plasmids are continuing to evolve or that tetM genes are being introduced into different families of 24.5 MDa conjugative plasmids.

Cephalosporins↗
PubMed2006

Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.

Animals↗
PubMed1977

Mutations of temperature sensitivity in R plasmid pSC101.

Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43 degrees C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 43 degrees C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells haboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells haboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotrophic phenotype, such as an increase of drug resistance level at 30 degrees C and a decrease in the number of plasmid genomes in a cell.

DNA, Bacterial↗
PubMed1982

Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology.

The frequency of plasmid establishment in the transformation of Streptococcus pneumoniae by plasmid DNA was increased more than 10-fold when the plasmid carried DNA homologous to the host chromosome. Perfect homology was not necessary for such facilitation; small additions or deletions were tolerated, but extensive deletions in the homologous segment of either plasmid or chromosome reduced or eliminated facilitation. The facilitated plasmid transfer showed a linear dependence on monomeric plasmid concentration rather than the quadratic dependence found in the absence of homology, which indicated that entering plasmid fragments interacted with the chromosome rather than with each other to establish a plasmid replicon. Restriction enzyme cleavage of the plasmid in the nonhomologous segment destroyed its activity, but cleavage in the homologous segment or even enzymatic removal of part of that segment did not prevent plasmid transfer, and plasmids of the original size were established. In facilitated transfer, chromosomal markers (additions and deletions as well as single-site mutations) entered the plasmid with a frequency ranging from 10 to 90% depending on the marker location. Several possible mechanisms for the establishment of plasmids in the presence of chromosomal homology and for the transfer of chromosomal information are considered. They depend on synapsis of the newly entered single-strand plasmid fragment with the host chromosome and subsequent copying of, donation from, or integration into the homologous chromosomal segment. After plasmid establishment, equilibration of donor and chromosomal markers between the chromosome and the plasmid pool, presumably by homologous recombination events, was observed.

Alleles↗
PubMed1997

Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2.

A 3.2-kb region of the broad-host-range plasmid RK2 has been shown to encode a highly efficient plasmid maintenance system that functions in a vector-independent manner. This region, designated par, consists of two divergently arranged operons: parCBA and parDE. The 0.7-kb parDE operon promotes plasmid stability by a postsegregational killing mechanism that ensures that plasmid-free daughter cells do not survive after cell division. The 2.3-kb parCBA operon encodes a site-specific resolvase protein (ParA) and its multimer resolution site (res) and two proteins (ParB and ParC) whose functions are as yet unknown. It has been proposed that the parCBA operon encodes a plasmid partitioning system (M. Gerlitz, O. Hrabak, and H. Schwabb, J. Bacteriol. 172:6194-6203, 1990; R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). To further define the role of this region in promoting the stable maintenance of plasmid RK2, the parCBA and parDE operons separately and the intact (parCBA/DE) par region (3.2 kb) were reintroduced into an RK2 plasmid deleted for par and assayed for plasmid stability in two Escherichia coli strains (MC1061K and MV10delta lac). The intact 3.2-kb region provided the highest degree of stability in the two strains tested. The ability of the parCBA or parDE region alone to promote stable maintenance in the E. coli strains was dependent on the particular strain and the growth temperature. Furthermore, the insertion of the ColE1 cer site into the RK2 plasmid deleted for the par region failed to stabilize the plasmid in the MC1061K strain, indicating that the multimer resolution activity encoded by parCBA is not by itself responsible for the stabilization activity observed for this operon. To examine the relative contributions of postsegregational cell killing and a possible partitioning function encoded by the intact 3.2-kb par region, stability assays were carried out with ParD provided in trans by a compatible (R6K) minireplicon to prevent postsegregational killing. In E. coli MV10delta lac, postsegregational killing appeared to be the predominant mechanism for stabilization since the presence of ParD substantially reduced the stability of plasmids carrying either the 3.2- or 0.7-kb region. However, in the case of E. coli MC1061K, the presence of ParD in trans did not result in a significant loss of stabilization by the 3.2-kb region, indicating that the putative partitioning function was largely responsible for RK2 maintenance. To examine the basis for the apparent differences in postsegregational killing between the two E. coli strains, transformation assays were carried out to determine the relative sensitivities of the strains to the ParE toxin protein. Consistent with the relatively small contribution of the postsegregational killing to plasmid stabilization in MC1061K, we found that this strain was substantially more resistant to killing by ParE in comparison to E. coli MV10delta lac. A transfer-deficient mutant of thepar-deleted plasmid was constructed for the stable maintenance studies. This plasmid was found to be lost from E. coli MV10delta lac at a rate three times greater than the rate for the transfer-proficient plasmid, suggesting that conjugation can also play a significant role in the maintenance of plasmid RK2.

Bacterial Proteins↗
PubMed1995

Natural plasmids of filamentous fungi.

Among eukaryotes, plasmids have been found in fungi and plants but not in animals. Most plasmids are mitochondrial. In filamentous fungi, plasmids are commonly encountered in isolates from natural populations. Individual populations may show a predominance of one type, but some plasmids have a global distribution, often crossing species boundaries. Surveys have shown that strains can contain more than one type of plasmid and that different types appear to be distributed independently. In crosses, plasmids are generally inherited maternally. Horizontal transmission is by cell contact. Circular plasmids are common only in Neurospora spp., but linear plasmids have been found in many fungi. Circular plasmids have one open reading frame (ORF) coding for a DNA polymerase or a reverse transcriptase. Linear plasmids generally have two ORFs, coding for presumptive DNA and RNA polymerases with amino acid motifs showing homology to viral polymerases. Plasmids often attain a high copy number, in excess of that of mitochondrial DNA. Linear plasmids have a protein attached to their 5' end, and this is presumed to act as a replication primer. Most plasmids are neutral passengers, but several linear plasmids integrate into mitochondrial DNA, causing death of the host culture. Inferred amino acid sequences of linear plasmid ORFs have been used to plot phylogenetic trees, which show a fair concordance with conventional trees. The circular Neurospora plasmids have replication systems that seem to be evolutionary intermediates between the RNA and the DNA worlds.

Ascomycota↗
PubMed1989

Genetic and molecular characterization of pR351 plasmid from enteropathogenic Escherichia coli strain.

A pR351 plasmid (Tc Ap Cb) conferring drug resistance of naturally occurring E. coli strain was examined. Conjugation and transduction experiments have indicated that this plasmid is R plasmid aggregate consisting of three independent plasmids: a) conjugative plasmid pR351 A (SuTc) fi- (F) belonging to incompatibility group L, b) conjugative plasmid pR351 B (SuApCb) fi- (F) belonging to incompatibility O, and c) non-conjugative plasmid pR351 C (ApCb). The existance of these plasmids in pR351 plasmid aggregate was confirmed by the agarose gel analysis of plasmid DNA isolated from the lysates of E. coli J53 transconjugants carrying pR351 A or pR351 B plasmids and from E. coli K12 C600 transductant carrying pR351 C plasmid. Molecular mass of these plasmids was found to be 55.60 and 3 Mdal respectively. The non-conjugative pR351 C plasmid could be mobilized by Col B and F factors. Our findings have indicated that two fi- (F) R plasmids can stably coexist in R plasmid aggregate.

Drug Resistance, Microbial↗
PubMed1983

Genetic properties of conjugative R plasmids in Escherichia coli and Salmonella isolated from feral and domestic pigeons, crows and kites.

A total of 62 conjugative R plasmids detected in 48 Escherichia coli and 19 conjugative R plasmids in 16 Salmonella strains from domestic (carrier and emerald) and feral pigeons, crows (Japanese jungle crows and carrion crows) and black kite, were examined for genetic properties. Out of the 62 E. coli R plasmids examined for fertility inhibition (Fi), 29 (46.8%) were Fi+, and the remainder were Fi-. Among the 62 R plasmids, 16 (25.8%) were classified into incompatibility (Inc) group FII, 15 (24.2%) into I alpha, 7 (11.3%) into H1 and 2 (3.2%) into N. The remaining 23 R plasmids could not be classified in this study. Among the 19 Salmonella R plasmids, 4 showed thermosensitive (ts) transfer. Out of the 18 plasmids tested, 13 (72.2%) including 4 ts-R plasmids were Fi-, and the remaining 5 (27.8%) were Fi+. Five R plasmids containing 4 Fi- were IncI alpha, while 4 ts-R plasmids belonged to IncH1 (1 R plasmid) and H2 (3 R plasmids). The remainings were not tested. In E. coli strains, IncFII and I alpha were widely (4 of 6 lots of birds, respectively) distributed, followed by N (2 lots) and H1 (1 lot). IncN R plasmid was detected from domestic pigeons and crows, but not from feral pigeons. In Salmonella strains from 5 lots of birds, IncFII and N plasmids which were detected from E. coli were not found, but IncH2 was detected from domestic pigeons (1 lot) and kite (1 lot). IncI alpha was detected from feral pigeons (1 lot) and kite (1 lot). The potential transfer of R plasmids between Salmonella and E. coli, and of dissemination of the plasmids among the free-flying birds, animals and humans is discussed.

Animals↗
PubMed1995

Competition between parental and recombinant plasmids affects the measure of recombination frequencies.

Recombination frequencies in multicopy plasmids are generally deduced from the rate of appearance of cells expressing a recombinant phenotype (i.e., "recombinant cells"). Detection of these cells requires not only the formation of a recombinant molecule but also the establishment of this molecule in the presence of the resident incompatible parental plasmid. Differences in fitness between parental and recombinant molecules will affect this establishment and could have great consequences for plasmid recombination measurements. To test this hypothesis, we compared recombination frequencies when the recombinant plasmid has or does not have a replication advantage over the parental plasmid. We used pBR322-derived plasmids which carry or lack the replication terminator TerB; recombination took place between directly repeated sequences of 16 bp and deleted TerB from the plasmid. The rate of appearance of recombinant cells strongly increased when the Tus/Ter system was active; however, we found no evidence for direct stimulation of recombination between direct repeats by replication fork stalling. The main factor responsible for the increase in the rate of appearance of recombinant cells when the parental plasmid carries TerB is the facilitated establishment of the recombinant plasmid since: (i) the transformation efficiency of the recombinant plasmid is higher in cells containing the Ter+ parent than in cells containing the Ter- parent; (ii) most recombinant plasmids did not lead to the appearance of recombinant cells when pBR322 was was not blocked by Tus, whereas the presence of TerB allows the detection of most events; and (iii) decreasing the parental plasmid copy number without modification of the recombinant plasmid leads to an exponential increase in the rate of appearance of recombinant cells. Our results show that the level of competition between parent and recombinant plasmids can greatly affect plasmid recombination frequencies deduced from the measure of recombinant cells. This effect can be as high as several orders of magnitude.

Base Sequence↗
PubMed1988

Origin and direction of replication in mitochondrial plasmid DNAs of broad bean, Vicia faba.

Broad bean (Vicia faba) mitochondrial DNA (mtDNA) includes three circular plasmids: mt-plasmid 1 (1,704 ntp), mt-plasmid 2 (1,695 ntp) and mt-plasmid 3 (1,476 ntp). Partially replicated circular forms of these mt-plasmids have been observed in electron microscope preparations. Restriction enzymes that cleave either mt-plasmid 2 (but not mt-plasmids 1 and 3) or mt-plasmid 3 (but not mt-plasmids 1 and 2) were used to generate linear forms of partially replicated mt-plasmid 2 and mt-plasmid 3 molecules. Analyses of these linearized replicative intermediates, observed by electron microscopy, indicated that in both mt-plasmid 2 and mt-plasmid 3 replication originates at a specific location and proceeds in the same, single direction around the molecules. The replication origins of mt-plasmid 2 and mt-plasmid 3 map close to sequences that can fold into hairpin structures.

DNA Replication↗
PubMed2005

Identification and distribution of sequences having similarity to mitochondrial plasmids in mitochondrial genomes of filamentous fungi.

Mitochondrial plasmids are autonomously replicating genetic elements commonly associated with fungal and plant species. Analysis of several plant and fungal mitochondrial genomes has revealed regions that show significant homology to mitochondrial plasmids, suggesting that plasmids have had a long-term association with their mitochondrial hosts. To assess the degree to which plasmids have invaded fungal mitochondrial genomes, BLAST search parameters were modified to identify plasmid sequences within highly AT-rich mtDNAs, and output data were parsed by E value, score, and sequence complexity. High scoring hits were evaluated for the presence of shared repetitive elements and location within plasmids and mtDNAs. Our searches revealed multiple sites of sequence similarity to four distinct plasmids in the wild-type mtDNA of Neurospora crassa, which collectively comprise more than 2% of the mitochondrial genome. Regions of plasmid similarity were not restricted to plasmids known to be associated with senescence, indicating that all mt plasmids can potentially integrate into mitochondrial DNA. Unexpectedly, plasmid-related sequences were found to be clustered in regions that have disproportionately low numbers of PstI palindromic sequences, suggesting that these repetitive elements may play a role in eliminating foreign DNA. A separate class of GC-rich palindromes was identified that appear to be mobile, as indicated by their occurrence within regions of plasmid homology. Sites of sequence similarity to mitochondrial plasmids were also detected in other filamentous fungi, but to a lesser degree. The tools developed here will be useful in assessing the contribution plasmids have made to mitochondrial function and in understanding the co-evolution of mitochondrial plasmids and their hosts.

Base Pairing↗
PubMed1986

Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity.

rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.

Bacterial Proteins↗
PubMed1987

Effects of recombinant plasmid size on cellular processes in Escherichia coli.

The effects of recombinant plasmid size on cell growth and viability, plasmid copy number, and synthesis of plasmid-encoded protein were investigated in Escherichia coli using plasmid pUC8 and four recombinant derivatives containing inserts of Drosophila melanogaster DNA of 1.7-6.0 kb. Growth in log phase was unaffected by plasmid size, but as plasmid size increased, maximum cell density decreased and, with the largest plasmid, cell death was accelerated after the stationary phase was reached. There was also a correlation between increasing plasmid size and decreased viability at high ampicillin concentrations, resistance to which is conferred by the plasmids. These effects were shown not to be due to transcription or translation of Drosophila sequences carried on the recombinant plasmids. Cells harboring the largest plasmid, pBS5 (8.7 kb), fared poorly in competition with plasmid-free cells in mixed cultures, compared with cells harboring pUC8 (2.7 kb). In addition, pBS5 was harbored at significantly fewer copies per cell than pUC8 at all phases of growth and supported much less production of the plasmid-encoded protein, beta-lactamase, than did pUC8. The results suggest that recombinant plasmid size may be an important parameter in the optimization of large-scale production of plasmid-encoded proteins.

DNA, Recombinant↗
PubMed1991

Maintenance of pBR322-derived plasmids without functional RNAI.

pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.

Bacteriocin Plasmids↗