Antagonism of the B subunit of DNA gyrase eliminates plasmids pBR322 and pMG110 from Escherichia coli.

The constructed plasmid pBR322 and the native plasmid pMG110 were eliminated (cured) from growing Escherichia coli cells by the antagonism of the B subunit of the bacterial enzyme DNA gyrase. The antagonism may be by the growth of cells (i) at semipermissive temperatures in a bacterial mutant containing a thermolabile gyrase B subunit or (ii) at semipermissive concentrations of coumermycin A1, an antibiotic that specifically inhibits the B subunit of DNA gyrase. The kinetics of plasmid elimination indicate that plasmid loss occurs too rapidly to be explained solely by the faster growth of that plasmid-free bacteria and, therefore, represents interference with plasmid maintenance.     

Inverted repeats in the DNA of plasmid pCU1

Renaturable regions in the DNA strands of the N group plasmid pCU1 have been visualized as stem-loop structures by electron microscopy. Four such distinct structures are described, the smallest of which is within the loop of a larger one. The region of pCU1 in which these structures occur has several restriction sites. This and the availability of plasmid deletions and recombinants has permitted the mapping of these structures relative to one another and to the restriction and functional map of the plasmid. The replication and maintenance region of the plasmid is located within one of these stem-loop structures.     

Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5.

Large plasmids from Agrobacterium tumefaciens, Salmonella typhimurium, Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa were routinely and consistently isolated using a procedure which does not require ultracentrifugation but includes steps designed to separate large-plasmid DNA from the bacterial folded chromosome. It also selectively removes fragments of broken chromosome. A variety of large plasmids was readily visualized with agarose gel electorphoresis, including five between 70 and 85 megadaltons (Mdal) in size, six between 90 and 143 Mdal, one that was larger than 200 Mdal, and one that was larger than 300 Mdal. This isolation procedure allowed initial estimation of the molecular sizes of the two IncP2 plasmids, pMG1 and pMG5, which were 312 and 280 Mdal, respectively. A standard curve for size determination by gel electrophoresis including plasmids between 23 and 143 Mdal in size did not extrapolate linearly for plasmids of the 300-Mdal size range. Unique response of different plasmids to the isolation procedure included sensitivity of IncP1 plasmids to high pH and the co-isolation of a 20-Mdal "cryptic" plasmid in conjunction.     

Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA.

In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.     

Copy number mutants of the broad-host-range Streptomyces plasmid pMG200

pMG200, isolated from the bacteriocin-releasing strain Streptomyces chrysomallus, was further physically mapped. Variants of S. chrysomallus were isolated which inhibited the parental strain. Two types of plasmids, pMG210 and pMG220, were isolated from these variants, with copy numbers of 10-30 and 300, respectively, compared with 1-3 for pMG200. pMG210 is apparently physically identical to pMG200 but presumably differs at a level not detected by simple restriction mapping; pMG220 is deleted for 1.6 kb. Genes for thiostrepton and viomycin resistance were subcloned from pIJ364 on to pMG200 and a fragment containing the gene for nourseothricin resistance was subcloned on to pMG220. In this way nonessential sites were identified.     

Inverted DNA repeats: a source of eukaryotic genomic instability.

While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes. Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae. Furthermore, they induce homologous recombination. Replication plays a major role in the deletion formation. Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3). The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable. Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp). The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination. Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat. We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.     

Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events

Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.     

Recombination between short direct repeats in Streptomyces lavendulae plasmid DNA.

Streptomyces lavendulae S985 carried two plasmids, pSL1 and pSL2. pSL2 contained all of the pSL1 sequences plus a tandem duplication of 900 base pairs from a region of pSL1. Sequence analysis of the duplication junction suggested that the duplication occurred by recombination between short direct repeats of as little as 5 base pairs.     

Inverted repeats,stem-loops,and cruciforms: Significance for initiation of DNA replication

Inverted repeats occur nonrandomly in the DNA of most organisms. Stem-loops and cruciforms can form from inverted repeats. Such structures have been detected in pro- and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem-loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti-cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four-way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication. © 1996 Wiley-Liss, Inc.     

Lack of plasmids in Streptomyces hydrogenans

Abstract Wild-type cells of Streptomyces hydrogenans ATCC 19631, strain HY A₁, show a remarkable degree of genetic instability with regard to the biosynthesis of 17β-hydroxysteroid dehydrogenase. As plasmids might be responsible for this phenomenon we tried to detect plasmids in lysates of this microorganism. Streptomyces lividans , strain TK64 (pIJ916), was used as reference strain, containing a 19-kb plasmid with low abundancy. Whereas plasmid DNA could be shown in lysates of S. lividans TK64, no plasmid DNA was detectable in lysates of S. hydrogenans .     

链霉菌质粒的复制与进化

近年来, 随着大质粒提取和检测技术的发展, 尤其是高通量DNA测序技术的应用, 使得链霉菌大的环型质粒和线型质粒的研究取得了较快的进展。相比于研究透彻的细菌Theta型复制的质粒, 链霉菌Theta型质粒在复制区的结构、复制蛋白和调控蛋白作用的分子机理等方面具有多样性和新颖性。新鉴定的许多线型质粒的中心复制区表明中心复制的起始可以靠近端粒, 一个质粒也可以有2个以上的复制区。新分离的端粒序列显示端粒“折返”不是必需的, 而形成二级结构对于端粒复制是重要的。链霉菌环型和线型质粒的测序分析显示它们之间存在亲缘关系。环型质粒可以与噬菌体共整合, 实验证明它们在一定条件下可以相互转换。这些研究结果表明, 链霉菌环型、线型质粒和噬菌体从结构到功能到进化具有多样性、新颖性和亲缘关系。     

Inverted DNA repeats channel repair of distant double-strand breaks into chromatid fusions and chromosomal rearrangements

Inverted DNA repeats are known to cause genomic instabilities. Here we demonstrate that double-strand DNA breaks (DSBs) introduced a large distance from inverted repeats in the yeast (Saccharomyces cerevisiae) chromosome lead to a burst of genomic instability. Inverted repeats located as far as 21 kb from each other caused chromosome rearrangements in response to a single DSB. We demonstrate that the DSB initiates a pairing interaction between inverted repeats, resulting in the formation of large dicentric inverted dimers. Furthermore, we observed that propagation of cells containing inverted dimers led to gross chromosomal rearrangements, including translocations, truncations, and amplifications. Finally, our data suggest that break-induced replication is responsible for the formation of translocations resulting from anaphase breakage of inverted dimers. We propose a model explaining the formation of inverted dicentric dimers by intermolecular single-strand annealing (SSA) between inverted DNA repeats. According to this model, anaphase breakage of inverted dicentric dimers leads to gross chromosomal rearrangements (GCR). This "SSA-GCR" pathway is likely to be important in the repair of isochromatid breaks resulting from collapsed replication forks, certain types of radiation, or telomere aberrations that mimic isochromatid breaks.     

Inverted repeats in the long-terminal repeats of the wheat retrotransposon Wis 2-1A.

The wheat insertion sequence Wis 2-1A possesses all the structural features characteristic of retrotransposons. Its long-terminal repeats (LTRs) are unusually long (1,755 bp) compared with those of other retrotransposons. Sequence analysis revealed that they differ from each other by only six point mutations. They contain a few tandem direct repeats, which could be explained by slippage mechanisms during replication. Almost half (44%) of the length of the LTRs is occupied by hairpin structures, which may relate to their large size. Possible origins of these inverted repeats are proposed, including the insertion and imprecise excision of transposable elements and errors when the DNA replication intermediate switches RNA template during retrotransposon replication.     

Isolation and characterization of Streptomyces erythreus plasmids

Streptomyces erythreus strains were found to carry several plasmids of molecular weights ranging from about 2 X 10(6) Mr to 40 X 10(6) Mr. Restriction enzyme maps for the streptomycete plasmids pPC7 and pPC8 were constructed for the enzymes Bg/II, EcoRI, XbaI, HindIII, BamHI and SalI. The smaller, pPC8, plasmid appears to be a naturally occurring deletion variant of pPC7. These plasmids belong to the group of conjugative streptomycete plasmids.     

Inverted repeats as genetic elements for promoting DNA inverted duplication: implications in gene amplification

Inverted repeats are important genetic elements for genome instability. In the current study we have investigated the role of inverted repeats in a DNA rearrangement reaction using a linear DNA substrate. We show that linear DNA substrates with terminal inverted repeats can efficiently transform Escherichia coli. The transformation products contain circular inverted dimers in which the DNA sequences between terminal inverted repeats are duplicated. In contrast to the recombination/rearrangement product of circular DNA substrates, which is exclusively one particular form of the inverted dimer, the rearrangement products of the linear DNA substrate consist of two isomeric forms of the inverted dimer. Escherichia coli mutants defective in RecBCD exhibit much reduced transformation efficiency, suggesting a role for RecBCD in the protection rather than destruction of these linear DNA substrates. These results suggest a model in which inverted repeats near the ends of a double-strand break can be processed by a helicase/exonuclease to form hairpin caps. Processing of hairpin capped DNA intermediates can then yield inverted duplications. Linear DNA substrates containing terminal inverted repeats can also be converted into inverted dimers in COS cells, suggesting conservation of this type of genome instability from bacteria to mammalian cells.     

Stability of pUB110 and pUB110-derived plasmids in Bacillus sphaericus 2362

The segregational and structural stability of pUB110 and four derivatives of various insert size and location was examined in the mosquito pathogen Bacillus sphaericus 2362 grown under conditions relevant to use of the bacterium as a larval control agent. Plasmids pUB110 (4.5 kb), pLDT103 (7.6 kb), and pTST130 (6.5 kb) exhibited 95–100% segregational stability at growth temperatures of 28° C and 38° C and at generation times of 42 and 108 min during 40 generations in a chemostat. Plasmids pLDT117 (9.7 kb) and pTST112 (6.5 kb), which had deletions in the BA4 membrane-binding site of the plasmids, were almost as stable (92–99%) unter these conditions. However, under growth conditions that allowed sporulation in mosquito larval cadavers, in batch culture or in the chemostat, plasmids with BA4-region deletions exhibited only 77–88% (pLDT117) or 66–81% stability in the resulting spores. Whereas segregational instability was associated with interruption of the BA4 region of these plasmids, this instability was primarily associated with the sporulation phase of development rather than with vegetative growth. Structural instability was not detected. Correspondence to: A. Yousten     

Inverted terminal repeats and terminal proteins of the genomes of pneumococcal phages

The nucleotide (nt) sequence at the ends of the genomes of the Streptococcus pneumoniae phages Cp-5 and Cp-7 has been determined and compared with the corresponding sequence of phage Cp-1. The genomes of phages Cp-5 and Cp-7 have inverted terminal repeats (ITRs) 343 and 347 bp long, respectively. In Cp-1 DNA the ITR is 236 bp long and the following 116 bp are 93% homologous. Some regions within the ITRs are conserved in the three genomes although the complete sequence of the ITRs is no more conserved than the rest of their genomes. The chromatographic behavior of their tryptic peptides suggests that the terminal proteins (TPs) of at least two of the phages are similar and that the TPs of the three pneumococcal phages differ markedly from that of the Bacillus subtilis phage psi 29.