Inverted repeats in the DNA of Streptomyces plasmids pMG110 and pMG120

Summary Two cryptic plasmids pMG110 (10.5 kb) and pMG120 (14.5 kb) isolated from Streptomyces luteolutescens were cleaved by restriction endonucleases BglII, KpnI, and SalGI. A physical map was constructed for pMG110. After denaturation and intrastrand reannealing, two types of snap-back structures were identified by electron microscopy, differing in the size of the loop (type 1, 1 kb; type 2, 1.6 kb), whereas the stem of both structures was about 190 bp long. Stem-loop structures of similar size were also observed in pMG120. In rare cases, both types of elements were present on the same DNA molecule. The analysis of BglII- and KpnI-generated fragments allowed the localization of the elements at two alternative positions on the physical map of pMG110.     

DNA structures contributing to the instability of recombinant plasmids inStreptococcus thermophilus

Summary Bifunctional shuttle vector (pBN183) and recombinant plasmids (pDCO2 and pDCO3) carrying aStreptomyces cholesterol oxidase gene (choA) were deleted to varying degrees inStreptococcus thermophilus. Restriction mapping of the deleted plasmids led to the identification of deletion prone regions in the transforming DNAs. Sequence analysis revealed that direct repeats and hairpin structures occurred in these regions, suggesting that they are deleterious to the stability of plasmids inS. thermophilus.     

Effect of the potential triplex DNA region on the in vitro expression of bacterial beta-lactamase gene in superhelical recombinant plasmids.

The effect of a pyrimidine/purine-biased stretch which has the potential to form an unusual triplex DNA structure on gene expression has been analyzed by measuring the activity of beta-lactamase as a reporter gene in recombinant plasmids. The Escherichia coli transformant carrying the plasmid p7ERS which has a potential triplex DNA region expressed about twofold more beta-lactamase activity than that carrying the plasmid pUC19. Since the expression of beta-lactamase has been shown to be affected by template topology in vitro, this in vivo observation suggests that the inserted pyrimidine/purine-biased stretch modulates the topology of flanking regions by forming unusual DNA structure to keep the template at the superhelicity favorable for the expression of beta-lactamase.     

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 long repeats in bacterial genomes reveals alternative evolutionary mechanisms in Bacillus subtilis and other competent prokaryotes.

Prokaryotic genomes seem to be optimized toward compactness and have therefore been thought to lack long redundant DNA sequences. However, we identified a large number of long strict repeats in eight prokaryotic complete genomes and found that their density is negatively correlated with genome size. A detailed analysis of the long repeats present in the genome of Bacillus subtilis revealed a very strict constraint on the spatial distribution of repeats in this genome. We interpret this as the hallmark of selection processes leading to the addition of new genetic information. Such addition is independent of insertion sequences and relies on the nonspecific DNA uptake by the competent cell and its subsequent integration in the chromosome in a circular form through a Campbell-like mechanism. Similar patterns are found in other competent genomes of Gram-negative bacteria and Archaea, suggesting a similar evolutionary mechanism. The correlation of the spatial distribution of repeats and the absence of insertion sequences in a genome may indicate, in the framework of our model, that mechanisms aiming at their avoidance/elimination have been developed.     

Analysis of two marine metagenomes reveals the diversity of plasmids in oceanic environments

Plasmid diversity is still poorly understood in pelagic marine environments. Metagenomic approaches have the potential to reveal the genetic diversity of microbes actually present in an environment and the contribution of mobile genetic elements such as plasmids. By searching metagenomic datasets from flow cytometry-sorted coastal California seawater samples dominated by cyanobacteria (SynMeta) and from the Global Ocean Survey (GOS) putative marine plasmid sequences were identified as well as their possible hosts in the same samples. Based on conserved plasmid replication protein sequences predicted from the SynMeta metagenomes, PCR primers were designed for amplification of one plasmid family and used to confirm that metagenomic contigs of this family were derived from plasmids. These results suggest that the majority of plasmids in SynMeta metagenomes were small and cryptic, encoding mostly their own replication proteins. In contrast, probable plasmid sequences identified in the GOS dataset showed more complexity, consistent with a much more diverse microbial population, and included genes involved in plasmid transfer, mobilization, stability and partitioning. Phylogenetic trees were constructed based on common replication protein functional domains and, even within one replication domain family, substantial diversity was found within and between different samples. However, some replication protein domain families appear to be rare in the marine environment.     

Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.

Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes.     

Analysis of Tn5 inversion events inEscherichia coli plasmids

The ability of the bacterial transposon Tn5 to undergo sequence inversion in Rec⁺ Escherichia coli cells as a result of recombination between its duplicated IS50 elements was examined using specially designed plasmid constructs. Surprisingly, recombination events in the IS50 elements that led to crossover and therefore Tn5 inversion could be detected at a frequency of only 10^?5. This was approximately an order of magnitude lower than the frequency of IS50 recombination that led to conversion events (i.e. non-reciprocal recombination) without crossover, and at least two orders of magnitude lower than the frequency of intermolecular recombination between IS50 elements on two different plasmids. These rare conversion and inversion events in Tn5 appeared to be due to intramolecular recombination and not simply to multiple rounds of reciprocal crossing over, since the heterodimeric intermediates that would be generated during the latter process could be readily isolated but were shown to yield a completely different set of plasmid products upon resolution.     

The role of DNA repeats and associated secondary structures in genomic instability and neoplasia

Tumour-associated genetic changes frequently involve DNA translocation or deletion. Many of these events will have arisen from initial genomic damage, induced by either the activity of endogenous metabolic processes or from exposure to environmental genotoxic agents. Although initial genomic damage will have been widely distributed, tumorigenic events are confined to certain DNA target sites. Furthermore, within these target sites there appear to be regions of preferential DNA rearrangement, and examination of these sites implies that the location and extent of such rearrangement may be influenced by DNA primary and secondary structure rather than simply by the point of damage. We selectively review evidence relating to DNA structures that may predispose certain regions of the genome to damage-induced rearrangement, and discuss the possible role of interstitial, inverted telomere-like sequence arrays in promoting chromosomal events of a type known to be associated with some human and animal tumours.     

An automated microplate-based method for monitoring DNA strand breaks in plasmids and bacterial artificial chromosomes

A method is described for high-throughput monitoring of DNA backbone integrity in plasmids and artificial chromosomes in solution. The method is based on the denaturation properties of double-stranded DNA in alkaline conditions and uses PicoGreen fluorochrome to monitor denaturation. In the present method, fluorescence enhancement of PicoGreen at pH 12.4 is normalised by its value at pH 8 to give a ratio that is proportional to the average backbone integrity of the DNA molecules in the sample. A good regression fit (r² > 0.98) was obtained when results derived from the present method and those derived from agarose gel electrophoresis were compared. Spiking experiments indicated that the method is sensitive enough to detect a proportion of 6% (v/v) molecules with an average of less than two breaks per molecule. Under manual operation, validation parameters such as inter-assay and intra-assay variation gave values of <5% coefficient of variation. Automation of the method showed equivalence to the manual procedure with high reproducibility and low variability within wells. The method described requires as little as 0.5 ng of DNA per well and a 96-well microplate can be analysed in 12 min providing an attractive option for analysis of high molecular weight vectors. A preparation of a 116 kb bacterial artificial chromosome was subjected to chemical and shear degradation and DNA integrity was tested using the method. Good correlation was obtained between time of chemical degradation and shear rate with fluorescence response. Results obtained from pulsed- field electrophoresis of sheared samples were in agreement with those obtained using the microplate-based method.     

Identification of an Rts1 DNA fragment conferring temperature-dependent instability to vector plasmids

The multiphenotypic drug resistance factor Rts1 expresses a temperature-dependent instability characteristic. This plasmid was digested with the restriction enzyme BamHI. A DNA fragment with a molecular weight of 5.6 MDa (the H fragment) was inserted into plasmid pBR322 (pFK896) or into pSC105 (pYH156) at the BamHI site. These plasmids were unstable at 42 degrees C but stable at 32 degrees C. A restriction-enzyme map of the H fragment was constructed and the instability phenotype (Tdi) was localized to a DNA fragment with 0.5 MDa molecular weight. The temperature-dependent loss of the unstable plasmid pFK896 is abrupt and no gradual plasmid loss of this multicopy recombinant plasmid is observed. The possibility that the Tdi phenotype is due to overgrowth of R- cells was eliminated.     

Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker

Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms.     

CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro.

A large increase in the length of a CGG tandem array is associated with a number of triplet expansion diseases, including fragile X syndrome, the most common cause of heritable mental retardation in humans. Expansion results in the appearance of a fragile site on the X chromosome in the region of the CGG array. We show here that CGG repeats readily form a series of barriers to DNA synthesis in vitro. There barriers form only when the (CGG)n strand is used as the template, are K(+)-dependent, template concentration-independent, and involve hydrogen bonding between guanines. Chemical modification experiments suggest these blocks to DNA synthesis result from the formation of a series of intrastrand tetraplexes. A number of lines of evidence suggest that both triplet expansion and chromosome fragility are the result of replication defects. Our data are discussed in the light of such evidence.     

Regulation of the replication of bacterial plasmids

This is a survey of the available data on the structure of the replication apparatus and the control mechanisms of bacterial plasmids replication. All plasmids are classified as having three types of replicons according to the mechanisms of replication initiation. The role of plasmid--determined positive and negative regulatory elements involved in controlling the number of copies and incompatibility is discussed.     

The origin of genetic instability in CCTG repeats

CCTG tetranucleotide repeat expansion is associated with a hereditary neurological disease called myotonic dystrophy type 2 (DM2). The underlying reasons that lead to genetic instability and thus repeat expansion during DNA replication remains elusive. Here, we have shown CCTG repeats have a high propensity to form metastable hairpin and dumbbell structures using high-resolution nuclear magnetic resonance (NMR) spectroscopy. When the repeat length is equal to three, a hairpin with a two-residue CT loop is formed. In addition to the hairpin, a dumbbell structure with two CT-loops is formed when the repeat length is equal to four. Nuclear Overhauser effect (NOE) and chemical shift data reveal both the hairpin and dumbbell structures contain a flexible stem comprising a C-bulge and a T·T mismatch. With the aid of single-site mutation samples, NMR results show these peculiar structures undergo dynamic conformational exchange. In addition to the intrinsic flexibility in the stem region of these structures, the exchange process also serves as an origin of genetic instability that leads to repeat expansion during DNA replication. The structural features provide important drug target information for developing therapeutics to inhibit the expansion process and thus the onset of DM2.