Replicon fusions promoted by the inverted repeats of Tn5. The right repeat is an insertion sequence.
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Bacteriophage lambda↗
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Predominant end-products of prophage Mu DNA transposition during the lytic cycle are replicon fusions.
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Bacteriophage mu↗
Relationship between DNA chain growth termination and replicon sizes in gamma-irradiated mouse cells.
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Animals↗
Role of the F factor oriV1 region in recA-independent illegitimate recombination. Stable replicon fusions of the F derivative pOX38 and pBR322-related plasmids.
We have used a mating protocol to isolate recA-independent recombinants of pOX38 , an F factor derivative, and the non-conjugative plasmid pMBO311 . Plasmid pMBO311 is a derivative of pBR322 carrying a DNA insertion that contains IS121 and shows no extensive sequence homology to pOX38 . Twenty-seven cointegrate molecules formed during independent mobilizations of pMBO311 by pOX38 were examined by restriction and Southern hybridization analysis. In general, there were two classes of recombinants. A minority class appears to have been mediated by IS121 , resulting in the formation of cointegrate molecules containing IS121 at the junctions between the two plasmids. The majority class (23/27) apparently involved reciprocal recombination between sites on pOX38 and pMBO311 . IS121 does not seem to be responsible for the formation of this type of cointegrate molecule, since similar structures were generated at approximately the same frequency during mobilization of control plasmids that do not contain IS121 . We have localized the regions involved in this second class of recombination events and find that most (17/23) occur at or near oriV1 , the primary replication initiation site of pOX38 . Twelve of the cointegrate molecules showed identical restriction and Southern hybridization patterns demonstrating a preferred region on pMBO311 as well. This site was localized just distal to the tet genes, within a 640-base AvaI-PvuII segment in the pBR322 portion of the molecule.
DNA Restriction Enzymes↗
Incompatibility properties of Col E1 and pMB1 derivative plasmids: random replication of multicopy replicons.
The incompatibility properties of Col E1-like plasmids have been examined in Rec+ and RecA- bacteria. Two Col E1- (or two pMB1-) derivative plasmids coreplicated in the same clone for many cell doublings, irrespective of the rec genotype of host bacteria. Their kinetics of segregation were found to be consistent with models that assume a random choice of template molecule for each plasmid replication event, but with models based on a single (master) template molecule per cell. In contrast, minimal coreplication of a Col E1- and a pMB1-derivative plasmid occurred, with the latter type rapidly excluding the former. We suggest here that the pMB1 derivatives, pMB9 and pBR322, are less sensitive than Col E1 derivatives to the putative inhibitor that regulates plasmid replication, due to base sequence differences in their target for the inhibitor, and consider one mechanism whereby the duplication of Col E1-like plasmids might be regulated.
Bacterial Proteins↗
Bacteriophage Mu: a transposing replicon.
Mu DNA replication has been carried out in vitro on cellophane discs in the presence of dBUTP. If the DNA is sheared to 80 kb pieces, the Mu replicas band anomalously in CsCl gradients between hybrid and light DNA density positions. The intermediate density DNA comprises semiconservatively replicated Mu sequences, flanked by unreplicated light DNA. This and previous data are consistent with replication occurring within Mu boundaries. Both the synthesis of Mu sequences and the intermediate density DNA are abolished by protein synthesis inhibition in vivo just prior to lysis on cellophane discs. These observations indicate that at least some steps in bona fide Mu transposition-replication are being observed in vitro.
Bacteriophage mu↗
The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication.
Mutants in bacterial topoisomerase (topo) IV are deficient in chromosomal partitioning. To investigate the basis of this phenotype, we examined plasmid DNA topology in conditionally lethal topo IV mutants. We found that dimeric catenated plasmids accumulated in vivo after topo IV inhibition. The catenanes were supercoiled, contained from 2 to > 32 nodes, and were the products of DNA synthesis. Electron microscopy and recombination tests proved that the catenanes have the unique structure predicted for replication intermediates. These data provide strong evidence for a model in which unlinking of the double helix can occur in two stages during DNA replication and for the critical role of topo IV in the second stage. The interlocks in the catenanes appear to be sequestered from DNA gyrase, perhaps by compartmentalization in an enzyme complex dedicated to partitioning.
Cell Compartmentation↗
Prokaryotic and eukaryotic replicons.
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Animals↗
Control of replication of FII plasmids: comparison of the basic replicons and of the copB systems of plasmids R100 and R1.
The copy numbers of the FII plasmids R1 and R100 were determined in four different ways and found to be identical. Deletion of one of the copy number control genes, copB, together with its promoter gives rise to plasmid copy mutants with an increased copy number. The increase was found to be 8- and 3.5-fold for plasmids R1 and R100, respectively. These deletion derivatives were found to be extremely sensitive to the presence of CopB activity from their own parent plasmid but not to that of the other plasmid. Hence, the CopB protein and its target are plasmid-specific and not FII-group-specific. These results are consistent with the high degree of nonhomology between plasmids R1 and R100 in a 250-bp region covering the distal part of the copB gene and the repA promoter region, which contains the target for the CopB protein.
Base Sequence↗
Overproduction of proteins encoded by lambda replicon-integrated plasmid: effect of partial inactivation of cI repressor.
Composite plasmids containing the 7.2-kb fragment of bacteriophage lambda cI857cro27, essential for lambda DNA replication and its control, have been constructed. The plasmids contained, in addition, pBR322, a part of Co1E1, and the Escherichia coli pss gene, coding for phosphatidylserine synthase. When the plasmid-harboring cells were induced at different temperatures, the phosphatidylserine synthase production was maximum at 37 degrees C, and the specific activities at 37 degrees C continuously increased until the culture reached saturation. The results suggest that, when the cro gene is defective, only a transient temperature for inactivation of cI repressor provides both the increase in copy number of the plasmids and cellular activities that allow overproduction of the plasmid-encoded protein.
Bacteriophage lambda↗
Identification and nucleotide sequence of the minimal replicon of the low-copy-number plasmid pBS2.
The plasmid pBS2 has a low copy number and is endogenous to Bacillus subtilis. The replication of this plasmid depends on the function of most of the host's dna genes including dnaB, which is unique to B. subtilis and is required for both the initiation of chromosome replication and the DNA-membrane association. We have identified the region that is essential for the replication of pBS2 and determined the complete 2279-bp nucleotide sequence of this region. In this region, there are two stretches of sequence homologous to the 18-bp consensus sequence which commonly appears at the origin of replication of plasmids pUB110 and pC194. The entire region contains six sizable open reading frames. Two of them are probably translated. One open reading frame, designated ORF A, coding for 269 amino acids, has significant homology, in terms of amino acid sequence, with the open reading frame of the gene for the Rep U protein of plasmid pUB110. The similarities between pBS2 and other plasmids suggest that the pBS2 may also replicate as a rolling circle, which appears to be the salient feature of a mechanism of replication that is common to small plasmids in gram-positive bacteria.
Amino Acid Sequence↗
Is 'senescence' of diploid cells an ad hoc mechanism suppressing 'replicon' or not?
So-called 'limited life span' of diploid cells does not depend on their unresponsiveness to growth factors from the data obtained by complete protein-free culture of tumor cells. Limited proliferation (called limited life span) of diploid cells is one of the 'ad-hoc' negative regulatory mechanisms in animals, and can not be differentiated from other inhibitory mechanisms called 'tumor suppression' and 'terminal differentiation'. Metabolic imbalance induced by proliferation-dependent time-bomb mechanisms including infidelity of DNA repair is suggested to explain limited proliferation of cells. After maturation, autonomic progression of negative regulators in cells is induced by no prohibition of terminal differentiation, since organisms prepare no programs to stop development and differentiation. It is an attractive hypothesis that a proliferation-dependent time-bomb has been developed to control organogenesis for maturation and to determine body size. It is true that limited proliferation of cells can not explain longevity of individuals, although the so-called longevity genes play roles in ageing. Teleologically, longevity of individuals has been developed to produce genetic heterogeneity according to the selfish gene theory. This inter- and intra-species genetic heterogeneity increases the probability of selfish gene replication in germ cell line. After maturation and reproduction of DNA in germ cell line, individuals as vehicles for the DNA can be discarded by the 'selfish' genes.
Animals↗
Functional analysis of pSM19035-derived replicons in Bacillus subtilis.
We determined the effect of various Bacillus subtilis dna(Ts) mutations on pSM19035 replication. At non-permissive temperature, plasmid replication depends on the dnaB, dnaC, dnaG and dnaI initiation replication products, but does not require the dnaA function. DNA elongation is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in dna(Ts) mutants impaired in different subunits of the enzyme. Single-stranded plasmid replication intermediates, corresponding to the leading strand, accumulate in the dnaD strain. On the basis of electron microscopic analysis of replication intermediates, we confirmed that the plasmid replicates unidirectionally by a theta mechanism.
Bacillus subtilis↗
A broad-host-range cloning vector transposable to various replicons.
A system is described for the stable insertion of cloned DNA sequences into the chromosomes of Gram-negative bacteria. Two broad-host-range plasmids form the basis of the system: one (the "carrier") contains a transposable DNA sequence into which foreign DNA can be cloned; the second (the "helper") provides transposition functions in trans. Both plasmids can be readily transferred between Gram-negative bacteria by conjugation. Instability of the carrier allows enrichment for the products of transposition to the chromosome of the new host, following which the insertion can be stabilised by elimination of the helper. The system was successfully tested in Escherichia coli, Methylophilus methylotrophus and Pseudomonas aeruginosa, and the insertions were stable in each case (less than 0.02% loss per generation).
Chromosomes, Bacterial↗
The nucleotide sequence of a mitochondrial replicon from maize.
The 1913-bp maize mitochondrial (mt) plasmid was isolated from a suspension culture of a Black Mexican Sweet maize strain, cloned into M13mp vectors, and sequenced by a unidirectional progressive deletion method. The 1.9-kb extrachromosomal double-stranded circular DNA plasmid was found to contain regions of sequence which in other systems are known to be part of origins of replication (ori). This plasmid could be used as a carrier for chimeric genes and a molecular probe for replication.
Amino Acid Sequence↗
Excision and integration of a self-transmissible replicon of Streptomyces ambofaciens.
When Streptomyces ambofaciens OSF was crossed with the plasmid-free Streptomyces lividans TK24, almost all S. lividans exconjugants contained the free 11.1-kb plasmid pOS1. Southern hybridizations showed that pOS1 was derived from the integrated copy of previously recognized plasmid pSAM2 present in strain OSF. A shorter derivative of pOS1 was constructed carrying the tsr gene in a non-essential region, and this pOS7 plasmid was used in transformation experiments with protoplasts of S. ambofaciens ATCC23877 (containing pSAM2 only as an integrated sequence) and S. ambofaciens DSM40697 (devoid of pSAM2-related forms). In both cases, some clones carrying pOS7 in an integrated state were found. Integration into strain ATCC23877 was into the pre-existing integrated copy of pSAM2. In contrast, plasmid pOS7 integrated through specific plasmidic and chromosomal sites into strain DSM40697. Thus it is probable that pSAM2 integrates by interaction between preferred regions of the plasmid and host genomes.
Animals↗
Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome.
Recombinant DNA libraries generated in vitro should in theory contain all of the sequences of the genomes from which they are derived. However, the literature is dotted with reports of sequences that cannot be recovered, are under-represented, or are highly unstable. In particular, long palindromic nucleotide sequences of perfect or near-perfect symmetry are either lethal to the vector or suffer deletions or other rearrangements that remove symmetry [Collins, Cold Spring Harbor Symp. Quant. Biol. 45 (1981) 409-416; Collins et al., Gene 19 (1982) 139-146; Hagan and Warren, Gene 24 (1983) 317-326]. We report here that mutation of a single gene, namely sbcC, can overcome this inviability and allow for the stable propagation of a 571-bp nearly perfect palindrome in Escherichia coli. This has implications for the choice of strains used for the recovery and analysis of cloned nucleotide sequences.
Bacteriophage lambda↗
Characterization of gram-positive broad host-range plasmids carrying a thermophilic replicon.
The cryptic plasmid pBC1 (1.6 kb) isolated from Bacillus coagulans Zu1961 was genetically marked with the genes for chloramphenicol and ampicillin resistance (CmR and ApR) from the Escherichia coli plasmid pJH101. The recombinant vector obtained (pCP49, 7.0 kb) replicated and expressed CmR in B. subtilis and CmR and ApR in E. coli. Different shuttle vectors for Gram+ bacteria were also constructed by inserting pBC1 into the Staphylococcus aureus plasmid pC194. The smallest of these, pLM6 (2.8 kb), containing essentially pBC1 and the chloramphenicol acetyl transferase gene from pC194, replicated in B. subtilis at a copy number of 60. By electroporation, these plasmids were introduced and stably maintained in B. subtilis, B. amyloliquefaciens, S. aureus, S. carnosus and Lactobacillus reuteri.
Bacillus subtilis↗