Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.     

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

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

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences.     

Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle

Bovine papilloma virus (BPV) replicates as a multicopy nuclear plasmid in mouse fibroblasts. Using fluorescence activated cell sorting and mitotic selection procedures, we show that the replication of BPV occurs throughout S phase of the cell cycle and that replication is confined to S phase. After one round of chromosomal DNA replication, almost one quarter of BPV plasmids have replicated more than once, while a similar number of plasmids have not replicated at all. While multiple forms of BPV exist in the cell, all forms show the same pattern of replication. These results are consistent with a model in which BPV plasmids are chosen at random for replication throughout, and only during, S phase and support the view that the completion of S phase is a specifically activated event in the cell cycle rather than simply the end of one round of chromosomal DNA replication.     

Activity of the c-myc Replicator at an Ectopic Chromosomal Location

DNA replication starts at multiple discrete sites across the human chromosomal c-myc region, including two or more sites within 2.4 kb upstream of the c-myc gene. The corresponding 2.4-kb c-myc origin fragment confers autonomously replicating sequence (ARS) activity on plasmids, which specifically initiate replication in the origin fragment in vitro and in vivo. To test whether the region that displays plasmid replicator activity also acts as a chromosomal replicator, HeLa cell sublines that each contain a single copy of the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanked by selectable markers were constructed. A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfecting a donor plasmid containing the 2.4-kb c-myc origin fragment and FRT, along with a plasmid expressing the yeast FLP recombinase, into cells containing a chromosomal FRT acceptor site. The amount of short nascent DNA strands at the chromosomal acceptor site was quantitated before and after targeted integration of the origin fragment. Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nascent DNA relative to that at the unoccupied acceptor site and to that after the insertion of non-myc DNA. The abundance of nascent strands was greatest close to the c-myc insert of the integrated donor plasmid, and significant increases in nascent strand abundance were observed at sites flanking the insertion. These results provide biochemical and genetic evidence for the existence of chromosomal replicators in metazoan cells and are consistent with the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA.     

Epstein-Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells.

Some possible ways in which replication of plasmids containing the Epstein-Barr virus (EBV) plasmid maintenance origin, oriP, might be controlled were investigated. Virtually all plasmid molecules were found to replicate no more than once per cell cycle, whether replication was observed after stable introduction of the plasmids into cells by drug selection or during the first few cell divisions after introducing the DNA into cells. The presence in the cells of excess amounts of EBNA1, the only viral protein needed for oriP function, did not increase the number of oriP-replicated plasmids maintained by cells under selection. In the cell lines studied, EBNA1 and oriP seem to lack the capacity to override the cellular controls that limit DNA replication to one initiation event per DNA molecule per S phase. The multicopy status of EBV-derived, selectable plasmids appears to result from the initial uptake by cells of large numbers of plasmid molecules, the efficient maintenance of these plasmids, and the pressure of genetic selection against plasmid loss. Other unknown controls must be responsible for the amplification of EBV genomes soon after latent infection of cells.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences. Received: 13 January 1998 / Accepted: 10 June 1998     

Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.     

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.     

Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA.

Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.     

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144.     

Nonintegrated plasmid-chromosome complexes in Escherichia coli.

A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts. Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows. (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent. (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication. (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size. (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.     

Replication and Control of Circular Bacterial Plasmids

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can “sense” and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.     

Suppression of the thermosensitive replication phenotype of the derivative plasmid of pI9789::Tn552 in Staphylococcus aureus may involve integration of the plasmid into the host chromosome

Abstract Plasmid-chromosome co-integration was found to be the mechanism of choice to overcome thermosensitivity of replication of the plasmid pS1 in PS80d and RN4220 strains of Staphylococcus aureus . The integration of the plasmid was sometimes accompanied by deletion of a specific section of the plasmid pS1 in PS80d. Growth of bacteriophage on strains containing the integrated plasmid and the subsequent use of the phage in transduction gave transductants containing plasmids that had regained their replication thermosensitivity. These plasmids had not acquired any detectable chromosomal DNA. The 16-kb EcoRI fragment of the PS80d chromosome that hybridizes to pS1 is the target for recombination in many cases, but apparently other sites are also used. This fragment contains sequence homologous to parts of the transposon Tn552 and it is probable that site-specific recombination is involved in the integration. The possible mechanisms for the integrations and the deletions are discussed.     

Linear plasmids in plant mitochondria: Peaceful coexistences or malicious invasions?

Plant mitochondria contain small extrachromosomal DNAs in addition to a large and complex main mitochondrial genome. These molecules can be regarded as extrachromosomal replicons or plasmids, of which there are two forms, circular and linear. Linear mitochondrial plasmids are present in many fungi and in some plants, but they seem to be absent from most animal cells. They usually have a common structural feature, called an invertron, that is characterized by the presence of terminal inverted repeats and proteins covalently attached to their 5 termini. Linear mitochondrial plasmids possess one to six ORFs that can encode unknown proteins but often code for the DNA and RNA polymerases. Although the functions of most linear plasmids in plant mitochondria are unknown, some plasmids may be associated with mitochondrial genome rearrangements and may have phenotypic effects due to their integration into mitochondrial genome. The Brassica 11.6-kb plasmid, one of the linear mitochondrial plasmids in plants, shows a non-maternal inheritance, in contrast to mitochondrial genomes. The origin of these plasmids is still a mystery, but indirect evidence indicates the possibility of horizontal transfer from fungal mitochondria. In this review, the main features of these unique DNAs present in plant mitochondria are described.     

Plasmids isolated from marine sediment microbial communities contain replication and incompatibility regions unrelated to those of known plasmid groups.

Two hundred ninety-seven bacteria carrying plasmids that range in size from 5 to 250 kb were identified from more than 1,000 aerobic heterotrophic bacteria isolated from coastal California marine sediments. While some isolates contained numerous (three to five) small (5- to 10-kb) plasmids, the majority of the natural isolates typically contained one large (40- to 100-kb) plasmid. By the method of plasmid isolation used in this study, the frequency of plasmid incidence ranged from 24 to 28% depending on the samples examined. Diversity of the plasmids occurring in the marine sediment bacterial populations was examined at the molecular level by hybridization with 14 different DNA probes specific for the incompatibility and replication (inc/rep) regions of a number of well-characterized plasmid incompatibility groups (repB/O, FIA, FII, FIB, HI1, HI2, I1, L/M, X, N, P, Q, W, and U). Interestingly, we found no DNA homology between the plasmids isolated from the culturable bacterial population of marine sediments and the replicon probes specific for numerous incompatibility groups developed by Couturier et al. (M. F. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Our findings suggest that plasmids in marine sediment microbial communities contain novel, as-yet-uncharacterized, incompatibility and replication regions and that the present replicon typing system, based primarily on plasmids derived from clinical isolates, may not be representative of the plasmid diversity occurring in some marine environments. Since the vast majority of marine bacteria are not culturable under laboratory conditions, we also screened microbial community DNA for the presence of broad- and narrow-host-range plasmid replication sequences. Although the replication origin of the conjugally promiscuous broad-host-range plasmid RK2 (incP) was not detectable in any of the plasmid-containing culturable marine isolates, DNA extracted from the microbial community and amplified by PCR yielded a positive signal for RK2 oriV replication sequences. The strength of the signal suggests the presence of a low level of the incP replicon within the marine microbial community. In contrast, replication sequences specific for the narrow-host-range plasmid F were not detectable in DNA extracted from marine sediment microbial communities. With the possible exception of mercuric chloride, phenotypic analysis of the 297 plasmid-bearing isolates did not demonstrate a correlation between plasmid content and antibiotic or heavy metal resistance traits.     

Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeasts.

Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.     

Plasmid segregation into minicells is associated with membrane attachment and independent of plasmid replication.

The role of plasmid replication in the segregation of plasmids into Escherichia coli minicells was investigated with temperature-sensitive replication mutants derived from E. coli plasmids ColE1 and pSC101. For as long as six generations of growth, at permissive or nonpermissive temperatures (when greater than 80% of plasmid replication was inhibited), the same amount of previously 3H-labeled plasmid DNA segregated into minicells. Density gradient separations of wild-type and temperature-sensitive plasmid DNA from both replicons segregated into the minicells showed that about 20 to 25% was stably associated with the minicell membrane at both temperatures. Electron microscopy showed this DNA to consist of circular plasmid molecules attached to the minicell membrane. These combined findings suggest that segregation of plasmids into minicells and their association with the minicell membrane are interrelated and independent of plasmid replication.