Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Plasmids”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,009 records · Page 56Linked to original sources
PubMed1987

Spiroplasma plasmids.

Extrachromosomal DNA, constituting plasmids or replicative forms of viruses, has been detected in a variety of spiroplasmas, particularly in Spiroplasma citri. Only a few of the S. citri plasmids were characterized by restriction enzyme mapping, and essentially nothing is known on functions encoded by the plasmids. Our studies revealed in S. citri (R8A2) an 8.0-kbp plasmid that differed from previously described plasmids in its restriction map. It was also clonable in pBR322. The plasmid, named pRA1, was found in large quantities as free plasmid in S. citri (R8A2) subclones of low passage level. In subclones of higher passage levels, free plasmid was replaced by plasmid sequences integrated into the spiroplasma chromosome, as revealed by Southern hybridization blots of digested spiroplasmal DNA with nick-translated pRA1 or its recombinant as probes. Significant quantities of integrated plasmid sequences were also observed in S. kunkelii and in Spiroplasma sp. P40. Small quantities of free and/or integrated plasmid DNA were detected in some spiroplasmas serologically and genotypically remote from S. citri. Chromosome-integrated pRA1 sequences were cloned into the Escherichia coli plasmids pUC13 and M13. Hybridization tests and restriction maps of these clones indicated that the integrated plasmid sequences consisted of small repetitive sequences inserted into specific sites on the spiroplasma chromosome. Despite the large number of the inserts they do not appear to affect significantly gene expression in the spiroplasma. Due to the abundance of free and integrated pRA1 in S. citri, nick-translated pRA1 was effective as a DNA probe in detecting small numbers of S. citri in infected periwinkle plants and leafhoppers.

Cloning, Molecular↗
PubMed1996

[Occurrence of virulent plasmids in strains of Yersinia obtained from various sources].

By the method of alkaline lysis the occurrence was studied of plasmid DNA in 122 strains of Yersinia. In 29 strains a single plasmid was found with characteristic growth of the organism on CRMOX (CRMOX+) medium. These features are encoded by genes located on the pYV virulence plasmid. The group of Yersinia strains possessing the virulence plasmid included 27 strains of Y. enterocolitica 0:3 isolated from clinical materials, among them 21 strains isolated from patients between January and September 1996, while the remaining two strains with the virulence plasmid belonged to Y. pseudotuberculosis antigenic group I. Following restriction analysis with the EcoRI enzyme of the virulence plasmids isolated from 27 strains of Y. enterocolitica 0:3 occurrence of only one restriction endonuclease pattern agreeing with the virulence plasmid pattern of Y. enterocolitica 0:3 strains reported in the literature was found in all strains. On the other hand, the restriction analysis of the virulence plasmids obtained from Y. enterocolitica 0:3 and Y. pseudotuberculosis I strains showed evident differences between these plasmids in the size of the particle and the restriction pattern. The plasmid size calculated on the basis of the analysis of the restriction endonuclease patterns for virulence plasmids isolated from the strains of Y. enterocolitica 0:3 and Y. pseudotuberculosis I was 71.5 thousands and 62.7 thousands of base pairs respectively, and was in the range 60-75 thousands bp (40-50 Mda) reported in the literature for virulence plasmids of Yersinia. The study showed that finding of characteristic growth of Yersinia organisms on the CRMOX medium can be useful for the detection of strains containing the pYV virulence plasmid and for the determination of their potential pathogenicity.

Plasmids↗
PubMed1977

Clonal distribution of resistance plasmid-carrying Salmonella typhimurium, mainly in the Middle East.

Strains of Salmonella typhimurium of predominantly Middle Eastern origin, but distributed from England to India, were found to carry at least three types of resistance plasmid. The most important was initially identified as an F(I) plasmid by compatibility tests, but differs from the F factor on the one hand and the F(I) factors R162 and ColV on the other. The three groups of F(I) plasmids can be distinguished by their compatibility reactions with the MP10 plasmid of S. typhimurium (Smith, Humphreys, Grindley, Grindley & Anderson, 1973) and group H(1) factors: the F factor is unilaterally incompatible with group H(1) (Smith, Grindley, Humphreys & Anderson, 1973; Anderson, 1975b); the F(I) factors are compatible with MP10 and group H(1); and F(I)me factors are incompatible with MP10 but compatible with H(1). The majority of S. typhimurium cultures belonged to phage type 208; most of those that did not, belonged to types related to 208. Only a minority of their F(I)me plasmids were autotransferring. The remainder were mobilizable by F-like plasmids, and by group H(1) and H(2) factors, but not by the fi(-) I(1) factor Delta, or by plasmids of the I(2), B, P, W, N and com 7 groups. The compatibility reactions of the autotransferring F(I)me plasmids were identical with those of the non-transferring members of the group, and both were large, single-copy plasmids.The S. typhimurium strains of this series carried A or AK, and SSu resistance determinants: small, probably multicopy, non-transferring plasmids similar to those originally described in phage type 29 of S. typhimurium (Anderson & Lewis, 1965b).These S. typhimurium cultures probably represent a clone of wide geographical distribution. The accurate epidemiological study of such clonal outbreaks requires, in addition to phage typing, precise identification of the plasmids harboured by the epidemic strains, and may have to be carried to the molecular level.F(I)me plasmids were identified in other drug-resistant salmonellas, notably in a strain of S. wien which caused large outbreaks of mainly paediatric infection in Algeria, and also spread to Britain. An F(I)me plasmid was found in S. typhi phage type 44 from Algeria, in which the phage-restricting properties of the plasmid are responsible for the specificity of the type.

Ampicillin↗
PubMed1981

Change of drug resistance patterns and genetic properties of R plasmids in Salmonella typhimurium of bovine origin isolated from 1970 to 1979 in northern Japan.

A total of 321 Salmonella typhimurium strains of bovine origin obtained in northern Japan during the period 1970-1979 were tested for drug resistance and detection of conjugative R plasmids. Three hundred and eighteen (99.1%) of these strains were resistant to one or more drugs. The isolation frequently of multiply drug-resistant strains tended to increase year by year. Two hundred and thirty-seven (74.5%) of these resistant strains carried conjugative R plasmids. A total of 308 R plasmids including 174 (56.5%) thermosensitive (ts) R plasmids were derived from the 237 drug-resistant strains, indicating that 71 (30.0%) strains have two different conjugative R plasmids in a single host cell. Of the 308 R plasmids examined for fertility inhibition (fi), 167 ts and 131 non-ts R plasmids were fi-. Of the 60 ts r plasmids examined for incompatibility, 50 were classified into H1 group and 10 into H2 group. Of the 52 non-ts R plasmids examined, 35 were classified into the I alpha-group and the remaining plasmids were untypable in our tests. Mercury resistance marker was found in about 20% of H1 R plasmids coding for multiresistance, and all of H2 R plasmids coded for resistance to tellurite. The clonal distribution of an S. typhimurium strain which carried an H1 R plasmid coding for resistance to six drugs and mercury was recognized in 1978 and 1979.

Animals↗
PubMed1985

Transfer of plasmid-borne aminoglycoside-resistance determinants in staphylococci.

Aminoglycoside-resistance determinants in staphylococci are borne on conjugative and non-conjugative plasmids. The conjugative plasmids were found in methicillin-resistant strains of Staphylococcus aureus isolated recently in Darwin and Sydney, Australia and in Houston, Texas, USA. These plasmids and the class-2 conjugative plasmid reported by Archer and Johnston (1983) had similar patterns of EcoR1 restriction-endonuclease fragments, encoded resistance to gentamicin, kanamycin and neomycin, transferred to a non-lysogenic recipient in conditions that promoted close cell-to-cell contact and mobilised a small, non-conjugative plasmid. A further plasmid, pWG14, encoding resistance to kanamycin, neomycin, streptomycin, erythromycin and lincomycin, also displayed conjugative properties but did not mobilise the small, non-conjugative plasmid. The transfer frequency of all conjugative plasmids was stimulated by the addition of polyethylene glycol, particularly at concentrations above 20%, to mixtures of donor and recipient broth cultures. Polyethylene glycol appeared to promote close cell-to-cell contact between donor and recipient cells. A representative of the most common aminoglycoside-resistance plasmids in Australian isolates of methicillin-resistant S. aureus was non-conjugative and transferred by a bacteriophage-mediated system to a lysogenic recipient. With the exception of plasmid pWG14, the conjugative plasmids were also transferred by a bacteriophage-mediated system. Furthermore, cultural conditions that favoured conjugative transfer of plasmids inhibited bacteriophage-mediated transfer and vice versa. The efficacy of the two transfer systems for analysing the plasmids of gentamicin-resistant, methicillin-resistant isolates of S. aureus has been compared.

Aminoglycosides↗
PubMed1985

Relatedness of tetracycline resistance plasmids among species of coagulase-negative staphylococci.

Four isolates of Staphylococcus aureus and 98 isolates of coagulase-negative staphylococci representing six species all obtained from endocervical cultures were examined for antimicrobial susceptibility and for the presence of plasmids. More than 80% of the isolates were susceptible to each of 12 antimicrobial agents tested, whereas only 33% were susceptible to penicillin G, 30% were susceptible to cadmium chloride, and 41% were susceptible to tetracycline. Although no species-related susceptibility or plasmid patterns were detected, 77 isolates contained at least one plasmid and 43 contained a plasmid similar in mass to a 2.7-megadalton tetracycline resistance plasmid previously reported in staphylococci. Association of tetracycline resistance with plasmids of this size in four species was determined from curing experiments. No plasmids homologous with the tetracycline resistance locus of the Escherichia coli plasmid pBR322 were found among 11 isolates examined by DNA hybridization. Homology with a 2.7-megadalton plasmids (pRC701) from an endocervical isolate of S. aureus, however, was apparent for 2.7-megadalton plasmids harbored by six isolates as well as with larger plasmids harbored by three isolates. Restriction analysis revealed that pRC701 shared structural identity with two plasmids of a similar mass from two species of coagulase-negative staphylococci as well as with a previously characterized tetracycline resistance plasmid originating in S. aureus.

Base Sequence↗
PubMed1971

Effects of the recipient strain and ultraviolet irradiation on transduction kinetics of the penicillinase plasmid of Staphylococcus aureus.

When the penicillinase plasmid of Staphylococcus aureus PS 81(P(81))(T(81)) was transferred to its cured derivative of PS 81(N(P))(T(81)), there was a fivefold increase in the transduction frequency of penicillinase plasmid markers after ultraviolet (UV) irradiation of the phage instead of the expected decrease typical for plasmid-borne markers. These results were independent of the transducing phage, the donor, and the method of curing the recipient and were also obtained with a cured derivative of PS 80(PI(80)). With PS 52, a naturally occurring penicillin-sensitive strain, and a cured transductant of PS 52 as the recipients, typical plasmid kinetics were observed. The plasmid location of penicillinase plasmid markers in transductants was confirmed by their instability in ethidium bromide (EB). In a cross between isogenic plasmids (PI(258)penZ cad x PI(258)penI asa ero), transductants were doubly selected for cadmium and erythromycin resistances. There was a twofold increase in transduction frequency after UV irradiation of the transducing phage and an increase in the proportion of recombinant type transductants. CsCl-EB density centrifugation revealed that plasmid deoxyribonucleic acid (DNA) was present in PS 81(P(81))(N(T)) and its cured derivative [PS 81(N(P))(N(T))], but not in PS 52. Sucrose gradient analysis of plasmid DNA showed that the penicillinase plasmid of PS 81(P(81))(N(T)) was larger than the plasmid in its cured derivative. Thus, the cured derivative contains plasmid DNA which appears to recombine with the incoming plasmid, causing the rise in transduction frequency noted after UV irradiation of transducing phage.

Arsenic↗
PubMed1969

Naturally occurring penicillinase plasmids in Staphylococcus aureus.

A series of plasmids harbored by naturally occurring penicillin-resistant strains of Staphylococcus aureus were surveyed with a view toward exploring the variability in plasmid-linked marker patterns. Plasmids were transduced from their natural hosts to either of two plasmid-negative laboratory strains by selection for cadmium resistance, and the transductants were tested for all other markers previously found to be plasmid-linked. All of the strains that were able to serve as genetic donors to one of the two stock strains could donate cadmium and lead resistance as linked, plasmid-borne markers. Among the other plasmid markers, a wide variety of patterns was found, including four plasmids that did not carry the penicillinase determinant. Each of the 26 plasmids studied, including the latter 4, was found to belong to one of the two incompatibility sets of penicillinase plasmids previously identified. With the exception of the penicillinase-negative plasmids, which were found in both sets, all the plasmids of incompatibility set I directed the production of penicillinase type A; those belonging to set II directed either type A or type C. Those of set II without exception increased the sensitivity of their host strains to bismuth ion; those of set I carried determinants of bismuth resistance or did not affect the sensitivity of their host to this ion. No other perfect correlations between markers were encountered; in particular, there was no correlation between penicillinase serotype and the excretion of the enzyme. This finding allows the prediction that there is, in addition to all of the markers thus far identified, a plasmid-linked determinant of penicillinase excretion.

Cadmium↗
PubMed2002

Stress responses and replication of plasmids in bacterial cells.

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

Journal Article↗
PubMed1993

Instability of hybrid plasmids in Bacillus subtilis.

We studied the mechanisms responsible for instability of hybrid derivatives of staphylococcal R plasmids in B. subtilis cells. Four pBCH plasmids were constructed from staphylococcal chloramphenicol-resistance plasmid pC756 ligated with pBR322 or pUC8 vectors, and two pAEC plasmids contained pC756 plasmid, erm gene from S. aureus pE3692 and pBR322 plasmid vector. The level of instability of these recombinant plasmids was clearly related to their sizes, the larger derivatives were segregationally highly unstable. The four hybrid pBE plasmids constructed from erythromycin-resistant pE3692 plasmid or its deletion derivative and pBR322 vector were poorly maintained in B. subtilis rec+ strain in comparison with the B. subtilis (recE4) strain. Moreover, the absence in pBE-hybrids of a part of palA sequence led to considerable reduction of plasmid stability in B. subtilis cells. However, the clones of B. subtilis harbouring pBE plasmids integrated into the bacterial chromosome presented much higher stability of the plasmid erm gene than the clones maintaining autonomously replicating plasmids.

Bacillus subtilis↗
PubMed1977

Molecular cloning and in vitro transcription of Bacillus subtilis plasmid in Escherichia coli.

A composite plasmid (pAT2010) has been constructed in vitro from RSF2124 and Bacillus subtilis IFO3022 plasmid (pAT1060) by covalent joining of the two DNA molecules by means of Escherichia coli DNA ligase through the cohesive ends generated by restriction endonuclease RI (EcoRI) cleavage. The composite plasmid was selected by transformation of E. coliC600r-m- with the ligated mixture after enrichment for composite plasmid by preparative agarose gel electrophoresis, and plating of the transformants on a medium containing ampicillin and colicin E1. Treatment of the composite plasmid with EcoRI yielded two fragments corresponding to the linear forms of the parental plasmids. The composite plasmids replicated as biologically functionally units in E. coli, and expressed genetic information carried by RSF2124. In the presence of chloramphenicol, the composite plasmids continued to replicate and the copy number gradually increased. Such nature of replication in the presence of chloramphenicol is characteristic to RSF2124 derived from colicin E1 factor, and so it is suggested that the replicator of RSF2124 is functional in the composite plasmid. The composite plasmid was found to synthesize mRNA of B. subtilis plasmid in cell-free extracts of E. Coli, by hybridization of the mRNA to the original plasmid DNA of pAT1060.

Bacillus subtilis↗
PubMed1984

Molecular comparisons of plasmids isolated from colicinogenic strains of Escherichia coli.

The plasmid content of 14 colicinogenic strains of Escherichia coli has been examined. Four strains contained miniplasmids (1.2-2.0 kb). Small plasmids (4-7 kb) were detected in all the strains specifying group A colicins (colicins A, E1, E2, E3 and K; group I plasmids). Larger plasmids (55-130 kb) were detected in seven out of nine strains specifying group B colicins (colicins B, H, Ia, Ib, M, V and S1; group II plasmids). DNA-DNA hybridization with group II plasmids showed a wide variation in the degree of DNA sequence homology among its members. In contrast little (if any) DNA sequence homology was detected with the group I plasmids when the same group II plasmid DNAs were used as hybridization probes. The results of DNA-DNA hybridization studies with the two small group II plasmids (pcolG-CA46 and pcolD-CA23) suggested that these plasmids are equivalent to deleted forms of larger group II plasmids. Our hybridization data thus support the division of colicin plasmids into the two groups (I and II) that have been previously defined from genetic and physiological studies.

Bacteriocin Plasmids↗
PubMed1981

Identification and characterization of Col plasmids from classical colicin E-producing strains.

A series of transformants have been derived which carry the Col plasmids from 11 E-colicin-producing strains isolated by Fredericq or Hamon. The ColE plasmids identified included four of type E1, five of type E2, and two of a type designated E4 by Horak (Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 233:58-63, 1975). Strain K317 was shown to carry a ColE7 plasmid and a 2.7-megadalton (Md) ColE2imm plasmid which confers immunity to colicin E2 but not the ability to produce colicin. Other plasmids identified in the colicinogenic isolates were a 3.4-Md ColN plasmid and a 1.3-Md Col plasmid of unknown type. The ColE1 plasmids all continued replicating in cultures treated with chloramphenicol. Strains carrying the ColE4 or ColE3-CA38 plasmid exhibited partial sensitivity to their own colicins and exhibited an unusual clearing-zone morphology when overlaid on stabs of colicin E2- or E7-producing strains. Except for ColE1-K53, ColE1-K47, ColE2-CA42, and ColE7-K317, all of the ColE plasmids were found to be about 4.3 Md in size. ColE2-CA42 and ColE7-K317 are both about 3.9 Md and are the only two plasmids of the E2, E3, E4, and E7 types that did not yield a 0.39-Md deoxyribonucleic acid fragment as an EcoRI digestion product. The comparisons of the ColE plasmids suggest several structural and functional relationships among them.

Bacteriocin Plasmids↗
PubMed2024

Putative evolution of Myxococcus fulvus 124B02 plasmid pMF1 from a chromosomal segment in another Myxococcus species.

Myxobacteria or order Myxococcales (old nomenclature) or phylum Myxococcota (new terminology) are fascinating organisms well known for their diverse peculiar physiological, taxonomic, and genomic properties. Researchers have long sought to identify plasmids within these organisms, yet thus far, only two organisms from different families have been found to harbor a plasmid. This study delves into the putative evolution of one of these plasmids, i.e., pMF1 present in Myxococcus fulvus 124B02 in the suborder Cystobacterineae and family Myxococcaceae. Here, we first reannotated the pMF1 plasmid genome sequence and identified two additional open reading frames or putative genes which were not annotated until now. We further reported that all pMF1 plasmid genes depict homology with Myxococcus stipitatus CYD1 draft genome (contig 28) and a chromosomal segment of M. stipitatus DSM14675 in a syntenic manner, implying the presence of plasmid-like structure in M. stipitatus CYD1, integrated into its chromosome. To comprehend the relationship among these three species, we conducted phylogenetic analyses using 16S and concatenated housekeeping genes and genome-to-genome distance calculator (GGDC) analysis, which confirmed that M. stipitatus CYD1 is a distinct and novel species within the genus Myxococcus. Overall, this comparative genomic study sheds light on the putative emergence of the pMF1 plasmid from a common ancestor of closely related yet distinct species, M. stipitatus CYD1, possibly through the partition from its chromosome as a segment.IMPORTANCEMyxobacteria are not well known to have plasmids. Until now, only two organisms have been shown to have plasmids, raising a pertinent question about how these plasmids evolved randomly within the phylum Myxococcota. The study presented in this manuscript delves into the emergence of the pMF1 plasmid found in Myxococcus fulvus 124B02, a member of the suborder Cystobacterineae and family Myxococcaceae. Our research addresses this intriguing topic of plasmid identification and evolution within myxobacteria, which are a group of fascinating organisms that have garnered significant interest due to their diverse physiological, taxonomic, and genomic properties.

Plasmids↗
PubMed2002

DNA plasmid transmission in yeast is associated with specific sub-nuclear localisation during cell division.

Circular plasmids in yeast carrying only an origin of DNA replication (ARS) exhibit maternal inheritance bias (MIB) and are poorly transmitted from mother to daughter cell during division. A variety of different sequences that overcome MIB have been described, including centromeric sequences (CEN), telomere-associated repeats, silencer sequences and a specific system encoded by the endogenous 2 micron circle plasmid requiring the cis-acting locus STB and the proteins Rep1 and Rep2. In each case, DNA segregation between mother and daughter cells is dependent on DNA-protein interactions. Using plasmids carrying multiple copies of a lac repressor binding sequence, we have localised DNA molecules in the yeast nucleus using a green fluorescent protein (GFP)-lac repressor fusion protein. We compared GFP localised plasmids carrying a centromere sequence with plasmids based on 2 micron circle carrying or lacking the STB sequences required for their segregation. We show that GFP localised plasmid carrying the complete STB locus co-localises with the plasmid proteins Rep1 and Rep2 to discrete chromatin sites. These sites are distinct from both the telomeres and from sites of cohesin binding. Deletion of the region of STB essential for the stability of the plasmid, leads to a loss of plasmid association with chromatin, relocalisation of plasmids towards the nuclear periphery, and a decrease in the Rep1 protein associated with the plasmid. We conclude that specific plasmid localisation is likely to be important in the overcoming of MIB in yeast.

Cell Division↗
PubMed2002

Estimating the instability parameters of plasmid-bearing cells. I. Chemostat culture.

What determines the stability of plasmid-bearing cells in natural and laboratory conditions? In order to answer this question in a quantitative manner, we need tools allowing the estimation of parameters governing plasmid loss in different environments. In the present work, we have developed two methods for the estimation of the instability parameters of plasmid-bearing cells growing in chemostat. These instability parameters are: (i) selection coefficient (or cost of the plasmid)alpha and (ii) the probability of plasmid loss at cell division tau(0). We have found that generally selection coefficient alpha changes during elimination of plasmid-bearing cells due to changes in substrate concentration; hence, methods which assume constancy of alpha are intrinsically imprecise. Instead, one can estimate selection coefficient at the beginning and the end of cultivation when the substrate concentration is approximately constant. Applying developed techniques to two sets of experimental data, we have found that (i) the cost of the plasmid pBR322 depended on the dilution rate in chemostat and was higher at low dilutions; (ii) high levels of plasmid gene expression led to a high cost of the plasmid pPHL-7; (iii) the probability of plasmid loss was lower at high levels of plasmid gene expression and independent of the dilution rate. We have also discussed the application of our results to understanding the basic biology of bacterial plasmids.

Animals↗
PubMed1995

Use of continuous culture for the selection of plasmids with improved segregational stability.

In this report a method that enables the selection of stable plasmid variants and the isolation of DNA sequences that improve plasmid maintenance is described. The method is based on the principle that in populations of cells carrying derivatives of a plasmid that differ only in the level of segregational stability, when grown in a chemostat under conditions with selective pressure on the plasmid, cells that carry more stable plasmid variants will be enriched. We developed the system for Lactococcus lactis using segregationally unstable derivatives of the gram-positive theta plasmid pAM beta 1 as selection vectors. The results showed that the method is suitable for the enrichment, and subsequent purification, of three classes of plasmids with improved maintenance properties. The first class involved mutations in the pAM beta 1-derived selection plasmid. These mutations resulted in increased copy numbers, thereby rendering the plasmid segregationally more stable. The other two classes were based on the insertion of additional, stability promoting, sequences in the selection plasmids. We showed that these sequences can constitute either replication functions derived from another plasmid or functions directly involved in plasmid maintenance. The site-specific resolution function of pAM beta 1 was used as an example of the latter functions. We anticipate that the method described should have general applicability for the development of stable host-vector systems in bacteria.

Bacteriological Techniques↗
PubMed2002

Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences.

The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences. In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil. This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322. [(3)H]Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug. Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil. We have also found that berenil induces DNA degradation in plasmids containing the homopolymer. Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids. Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect. Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected.

DNA↗