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Two independent conjugal transfer systems operating in Bacteroides fragilis V479-1.

Bacteroides fragilis V479-1 (also designated strain 92) has previously been shown to contain a conjugative plasmid, pBF4, that specifies resistance to clindamycin (Cc). A report of inducible tetracycline (Tc) resistance in this strain suggested that this phenotype was also plasmid associated (G. Privitera et al., Nature [London] 278:657-659, 1979) and prompted further investigation. Mating experiments with V469-1 and a Cc-sensitive derivative of V479-1, V598, showed that Tc resistance transfer occurred by a conjugation-like event which was insensitive to DNase, was not mediated by donor culture cell-free filtrates, and required cell-to-cell contact. Results from transfer experiments with V479-1 indicated that Tc and Cc resistance determinants were not linked and segregated independently in matings. Progeny recovered from matings with the V479-1 or V598 donor strain were able to transfer the Tc resistance marker in secondary crosses. Tc resistance transfer from V479-1 or V598 was greatly stimulated by pregrowth in the presence of Tc but not Cc. pBF4-mediated Cc resistance transfer was not affected by pregrowth in the presence of Cc or Tc. Filter blot DNA hybridization studies revealed that pBF4 sequences were not involved in either the Tc resistant phenotype or its associated conjugal transfer properties. The Tc resistance transfer element was not associated with pBF4 or any other extrachromosomal DNA element.

Bacteroides↗

Comparative study of Epstein-Barr virus and SV40-based shuttle-expression vectors in human repair-deficient cells.

Shuttle vectors and expression vectors have been used in human cells to examine various aspects of DNA repair including effects of DNA damage on mutagenesis, transcription, replication and recombination. A combined shuttle-expression system should provide further advantages for the stable expression of and perhaps selection/rescue strategies for DNA repair genes. We describe 2 such systems. The first is a simian virus 40 (SV40) shuttle system which allows a quasi-stable episomal vector/host relationship in which the shuttle vector may be recovered in extrachromosomal DNA preparations many months after transfection and selection but in which a high proportion of the plasmids rescued in bacteria are heavily mutated and rearranged. Secondly, we describe Epstein-Barr virus-based shuttle-expression vectors which exist as stable, multicopy episomes in human cells. Using a reporter gene and a metal-inducible promoter we have obtained low basal and very high induced expression from episomal vectors in a variety of human cells including xeroderma pigmentosum and ataxia telangiectasia cell lines. This should facilitate many molecular genetic experiments in human cells and may have particular application to molecular cloning, expression and analysis of DNA repair genes.

Animals↗

Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations.

Plasmid profiles of 35 Lactobacillus plantarum strains isolated from different green olive fermentors were obtained. A large number of plasmids in the CCC form (from 5 to 16) were present in all the tested strains as confirmed by a second dimension electrophoresis of DNA. These plasmids, all of which remain cryptic, ranged from 2.0 to 68 kb in size. Novobiocin, sodium dodecyl sulphate and ethidium bromide were used as plasmid-curing agents but only novobiocin induced loss of extrachromosomal DNA at a high frequency in these strains.

DNA, Bacterial↗

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called 'opines', e.g. octopine (N-alpha-(D-1-carboxyethyl)-L-arginine) and nopaline (N-alpha-(1,3-dicarboxypropyl)-L-argine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a 'genetic colonization' of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.

DNA Restriction Enzymes↗

Production of antimicrobial substances, by hospital bacteria, active against other micro-organisms.

Fifty-nine clinical strains of bacteria, isolated from patients in the Regional Hospital of Talca, were studied. Seventy-four percent of these strains produced antibacterial substances, in comparison with 18% of the same bacterial species obtained from patients from a non-hospital habitat. Almost all the bacteria isolated from hospitalized patients demonstrated in vitro resistance to different antimicrobial agents. Pseudomonas aeruginosa was the most frequent species producing antibacterial substances and its products were of high potency, with a wide spectrum of antimicrobial activity. Cure of plasmid DNA, in most of the antibacterial-producer strains, resulted in the loss of their lethal activity and they also became susceptible in vitro to anti-microbials. These results indicated that such properties are encoded in extrachromosomal DNA. We believe that the knowledge of the antimicrobial activity and resistance to antimicrobials of bacteria from a hospital habitat can help explain the selection and persistence of such strains in this particular ecological niche.

Anti-Bacterial Agents↗

A system-level metastable model of cancer evolution: integrating replication stress, cell cycle deregulation and chromosomal instability.

INTRODUCTION: Cancer cell proliferation occurs within the context of persistent genomic instability. In this review, we propose the RS-CCD-CIN axis as a systems-level framework in which replication stress (RS), cell cycle deregulation (CCD) and chromosomal instability (CIN) form an interdependent triad that shapes tumour evolution. This axis represents a constrained metastable state in which genomic instability is tolerated and buffered. The objective of this review is to synthesize the current understanding of how the RS-CCD-CIN axis contributes to tumour heterogeneity, adaptability and therapy response. DISCUSSION: Evidence indicates that RS, CCD and CIN operate as a dynamic, interconnected network rather than as independent processes. Replication stress induces DNA damage and mutagenesis, while partial checkpoint disruption permits cells with unresolved lesions to proliferate. Chromosomal instability generates both structural and numerical alterations, contributing to intratumoural heterogeneity. Together, these processes facilitate adaptation to environmental and therapeutic pressures. Extrachromosomal DNA, micronuclei formation and cytosolic DNA signalling, including the cGAS-STING pathway, connect genomic instability to adaptive responses and immune modulation. Single-cell and spatial profiling reveal temporal and spatial variability in RS, CCD and CIN states, highlighting the limitations of static biomarkers. Therapeutically, targeting individual components often yields limited durability, whereas approaches that simultaneously perturb multiple aspects of the RS-CCD-CIN axis may improve clinical outcomes. CONCLUSIONS: This review highlights the RS-CCD-CIN axis as a fragile and metastable architecture that supports cancer evolution, while also being susceptible to collapse. A deeper understanding of this interconnected framework may inform the development of therapeutic strategies and enhance the management of resistance.

Humans↗

Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis.

A simple gel technique is described for the detection of large, covalently closed, circular DNA molecules in eucaryotic cells. The procedure is based on the electrophoretic technique of Eckhardt (T. Eckhardt, Plasmid 1:584-588, 1978) for detecting bacterial plasmids and has been modified for the detection of circular and linear extrachromosomal herpesvirus genomes in mammalian cells. Gentle lysis of suspended cells in the well of an agarose gel followed by high-voltage electrophoresis allows separation of extrachromosomal DNA from the bulk of cellular DNA. Circular viral DNA from cells which carry the genomes of Epstein-Barr virus, Herpesvirus saimiri, and Herpesvirus ateles can be detected in these gels as sharp bands which comigrate with bacterial plasmid DNA of 208 kilobases. Epstein-Barr virus producer cell lines also show a sharp band of linear 160-kilobase DNA. The kinetics of the appearance of this linear band after induction of viral replication after temperature shift parallels the known kinetics of Epstein-Barr virus production in these cell lines. Hybridization of DNA after transfer to filters shows that the circular and linear DNA bands are virus specific and that as little as 0.25 Epstein-Barr virus genome per cell can be detected. The technique is simple, rapid, and sensitive and requires relatively low amounts of cells (0.5 X 10(6) to 2.5 X 10(6)).

Animals↗

Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids.

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.

Animals↗

Engineering a mini-herpesvirus as a general strategy to transduce up to 180 kb of functional self-replicating human mini-chromosomes.

The engineering of therapeutic human artificial episomal chromosomes, HAECs, requires the development of strategies to deliver large functional self-replicating extrachromosomal DNA in target cells. Members of the herpesviral family are among the largest episomal double-stranded DNA viruses. As model systems of this family of endemic infectious agents, vectors derived from the human herpes 4 Epstein-Barr virus (EBV) were constructed which transferred up to 180 kb of DNA packaged as infectious virions. Such a transduction strategy was based on a non-oncogenic helper-dependent mini-EBV carrying minimal cis elements for latent replication and virus production. After exposure of human B lymphoma and lymphoblastoid cells to mini-EBVs transducing lacZ and human HPRT minigenes, stable cell transformants were selected which carried the delivered multimeric linear DNAs as circular episomes up to 160-180 kb in size. Following transduction of Lesch-Nyhan disease cells with a mini-EBV/HPRT, normal human HPRT function was restored in cells carrying large episomal HPRT minigenes. Direct visualization of the therapeutic mini-EBV by fluorescent in situ hybridization (FISH) on metaphase and interphase nuclei indicated that 99% (556/563) of the transduced mini-EBV DNA was episomal with an average copy number of one to two per nucleus. This system should allow the delivery of large genes in common diseases such as hemophilia A and codelivery of multiple genes in cells from polygenic diseases such as cancer. The extrachromosomal mini-EBV-based strategy offers an alternative to integrative or non-replicating gene therapy infectious vectors, which may be generally applicable to other herpesviruses characterized by different tropisms.

B-Lymphocytes↗

Identification of a DNA sequence associated with plasmid integration in Streptomyces coelicolor A3(2).

We identified a DNA element of length about 1 kb that is present in two copies in the chromosome of Streptomyces coelicolor A3(2) and is also present on the plasmid SCP1 which has been carefully defined genetically, but never isolated as extrachromosomal DNA. A copy of the element is close (within 5 kb) of a gene coding for an extracellular agarase in the chromosome of S. coelicolor A3(2) and in an NF strain, in which SCP1 has integrated into the chromosome, the agarase gene has been deleted. The element has properties reminiscent of Insertion Sequences in Escherichia coli, but it is not yet know if it can transpose.

DNA Restriction Enzymes↗

Survival mechanisms for Streptomyces linear replicons after telomere damage.

The ability of linear replicons to propagate their DNA after telomere damage is essential for perpetuation of the genetic information they carry. We introduced deletions at specific locations within telomeres of streptomycete linear plasmids and investigated mechanisms that enable survival. Here, we report that rescue of such plasmids in Streptomyces lividans occurs by three distinct types of events: (i) repair of the damaged telomere by homologous recombination; (ii) circularization of the plasmid by non-homologous end-to-end joining; and (iii) formation of long palindromic linear plasmids that duplicate the intact telomere by a non-recombinational process. The relative frequency of use of these survival mechanisms depended on the location and length of the telomeric DNA deletion. Repair by intermolecular recombination between the telomeres of chromosomes and plasmids, deletion of additional DNA during plasmid circularization, and insertion of chromosomal DNA fragments into plasmids during end-to-end joining were observed. Our results show that damage to telomeres of Streptomyces linear replicons can promote major structural transformations in these replicons as well as genetic exchange between chromosomes and extrachromosomal DNA. Our findings also suggest that spontaneous circularization of linear Streptomyces chromosomes may be a biological response to instances of telomere damage that cannot be repaired by homologous recombination.

Chromosomes, Bacterial↗

DNA-mediated alteration of the reversion frequency of transformed NIH/3T3 cells.

Cell selection immediately after DNA-mediated transfection of whole-cell DNA into mammalian cells has been used to select for specific DNA sequences that cause a phenotypic effect. Whole-cell mouse or human DNA was cleaved into a distribution of lengths (0.4-25 kilobase pairs) and transfected into anchorage-independent spontaneously transformed NIH/3T3 cells. Immediately after transaction, anchorage-dependent serum concentration-dependent reverents were selected. The Hirt supernatant, containing extrachromosomal DNA resulting from the transfection, was isolated from the revertants and transfected with high molecular weight carrier DNA into a second population of transformed cells; revertants were again selected. After five to seven cycles of transfection of Hirt supernatant DNA (obtained from revertants selected at the previous cycle) into new populations of transformed cells at each cycle, the reversion frequency had become 5-15 times greater than the spontaneous reversion frequency measured for several subclones of nontransfected or mocktransfected transformed NIH/3T3 cells. When nonmammalian genomic DNAs were used in transfecting a first population of cells, there was no effect on the reversion of frequency even after six cycles of selection. The reversion-enhancing activity of sixth-cycle Hirt supernatant DNA resulting after transfection at the first cycle with mouse or human sequences was destroyed by EcoRI but not by BamHI or Sal I. Sequences resembling human Alu I sequences were found in mouse whole-cell DNA isolated from sixth-cycle revertants generated after transfection of human sequences at the first cycle.

Animals↗

DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species.

DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.

Amino Acid Sequence↗

Human cells deficient in p53 regulated p21(waf1/cip1) expression exhibit normal nucleotide excision repair of UV-induced DNA damage.

Cancer development requires the accumulation of numerous genetic changes, which are believed to initiate through the presence of unrepaired lesions in the genome. In the absence of proficient repair, genotoxic agents can lead to crucial mutations of vital cellular genes via replication of damaged DNA. Many cell cycle regulatory proteins are known to modulate the repair capacity and consequently the fate of cells. We and others have recently shown that p53 tumor suppressor gene product is required for efficient global genomic repair (GGR) but not the transcription coupled repair (TCR) of the nucleotide excision repair (NER) sub-pathways. In order to discern the nature of the p53 modulation to be direct or indirect through a downstream mediator, we have investigated the processing of UV radiation induced lesions in human colon carcinoma, HCT116 cells expressing wild-type p53 but having different p21(waf1cip1) (hereafter p21) genotypes (p21+/+, p21+/-, p21-/-). Following 20 J/m(2) UV, all the three cell lines showed rapid increase in p53 protein but the accompanying increase in the expression of its downstream target protein p21 could only be seen in p21+/+ and p21+/- cells and not in p21-/- cells. Nevertheless, an absence of detectable p21 protein in deficient cells had no demonstrable effect on DNA repair response to UV irradiation, as measured by an immunoassay to detect removal of UV photoproducts from genomic DNA (GGR) and by individual strand specific removal of endonuclease-sensitive CPD from a target gene fragment (TCR). Introduction of cytomegalovirus (CMV)-driven luciferase reporter plasmid, UV damaged in vitro, into the un-irradiated cells of varying p21 background, revealed a relatively small but statistically significant decrease in the reporter expression in the host p21-/- as compared with p21+/+ and p21+/- HCT116 cells. Super-expression of p21 protein upon reintroduction of p21 expression construct, showed an enhanced recovery of UV damaged reporter activity that was not greatly different from a similar enhancement observed with undamaged plasmid reporter DNA. Taken together, the results indicate that (i) the p21 protein does not have a significant role in the repair of genomic DNA at chromosomal level; (ii) the well-established p53 dependent modulation of NER is distinct and independent of its cell cycle checkpoint function; and (iii) the reproducible enhancing effect of p21 expression observed through host cell reactivation (HCR) of extrachromosomal DNA is mainly attributable to an effect exerted on transcription rather than repair.

Cyclin-Dependent Kinase Inhibitor p21↗

Mutagenicity of a unique 8-oxoguanine in a human Ha-ras sequence in mammalian cells.

The processing of a unique 8-oxoguanine residue in DNA has been studied in mammalian cells using a single-stranded shuttle vector. A fragment of human Ha-ras carrying the lesion on the first (G1) or the second guanine (G2) of codon 12 was inserted in a shuttle plasmid. Extrachromosomal DNA is replicated in animal cells, extracted and used to transform bacteria to be amplified and individualized. DNA sequencing of bacterial clones showed the mutagenic potency of 8-oxoguanine in vivo to be approximately 4%. The presence of the 8-oxoguanine does not greatly affect survival of the progeny. No significant difference was observed between the mutation frequencies induced by 8-oxoguanine located either at the G1 or G2 position. The majority of the mutations, targeted at the lesion level, are G to T transversions. These base substitutions induced respectively glycine to cysteine (G1) or valine (G2) change in the P21ras protein. These mutations may contribute to activation of the protooncogene, leading to spontaneous tumorigenesis.

Animals↗

Genotypic characterization of seven strains of Mycoplasma fermentans isolated from synovial fluids of patients with arthritis.

We performed a genotypic characterization of seven strains of Mycoplasma fermentans which have been isolated from the synovial fluid of patients with rheumatoid arthritis (n = 2), spondyloarthropathy (n = 1), and unclassified arthritis (n = 4). We compared them to three reference strains (strains PG18 and K7 and incognitus strain) and to a clinical isolate from the urethra of a patient with nongonococcal urethritis. The characterization methods included electrophoresis of native DNA, arbitrarily primed PCR, and restriction fragment length polymorphism analysis following conventional and pulsed-field gel electrophoresis. Southern blot analysis with a probe internal to an insertion sequence was performed with the restriction products produced by the last two techniques. No extrachromosomal DNA sequences were detected. The M. fermentans strains identified by these methods did not present a unique profile, but they could be separated into two main categories: four articular isolates were genetically related to PG18 and the three other isolates, the urethral isolate, and the incognitus strain were related to K7. We also looked for the presence of the bacteriophage MAV1 (associated with the arthritogenic property of Mycoplasma arthritidis in rodents) in the M. fermentans strains. MAV1 DNA was not detected in either the clinical isolates or the reference strains of M. fermentans.

Adult↗

[Identification and characteristics of plasmids of the strains of erythromycin-producing Streptomyces erythreus].

Presence of plasmid DNA was investigated in laboratory strains 2 and 4 (NRRL 2338) of S. erythreus, as well as in strains 1 and 3 of S. erythreus subjected to improvement with respect to erythromycin production. Families of plasmids close by their molecular weights were identified in S. erythreus strains 3 and 4 (NRRL 2338). A plasmid DNA fraction of S. erythreus strain 3 was studied with electron microscopy. It enabled to identify 5 plasmids: pSE11, pSE12, pSE13, pSE14 and pSE15 with length of 5.3, 12.4, 16.3, 29.6 and 86.9 kb respectively. Using of various procedures for isolation of extrachromosomal DNA did not provide its detection in S. erythreus strains 1 and 2. At least a part of the plasmids detected in S. erythreus strains 3 and 4 (NRRL 2338) was conjugative. 32R-Labeled plasmid DNA of S. erythreus strain 3 was subjected to hydridization according to Sauthern with total DNA of the 4 strains treated with restrictases BamHI, PstI and BgIII. The studies showed that the genome of S. erythreus strain 2 was not homologous with the probe while S. erythreus strain 1 contained one of the plasmids or its part in chromosome-integrated state. In strains 3 and 4 (NRRL 2338) of S. erythreus certain plasmid DNAs were present in both autonomous and chromosome-inserted states. 32P-Labeled gene of erythromycin resistance (ermE) was subjected to hybridization according to Southern with total DNA of the 4 strains and with DNA plasmid fraction of S. erythreus strain 3. The signal was positive only in hydridization of the probe with total DNA of S. erythreus strains 1, 3, and 4 (NRRL 2338).(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Restriction Enzymes↗

[Establishment and use of infectious bacterial artificial chromosome (BAC) DNA clones of animal herpesviruses].

Since the first description of the cloning und mutagenesis of a herpesviral genome in 1997, a number of herpesviral DNA's have been cloned as bacterial artificial chromosomes (BACs). Herpesvirus BAC's are established by introduction of a mini F origin of replication into the viral genome, which can as such be maintained und manipulated in Escherichia coli as a single copy extrachromosomal DNA. Herpesvirus DNA's cloned as BAC's are accessible to the DNA repair und mutagenesis apparatus encoded by E. coli, which can be exploited for the generation und subsequent analysis of virus mutants. One of the advantages of BAC cloning und mutagenesis is that it is independent of virus growth in cultured cells, thereby minimizing the risk of compensatory mutations. Here we summarize BAC's that have been established for Herpesviruses of livestock, among them Bovine Herpesvirus Type 1 (BHV-1), Equine Herpesvirus Type 1 (EHV-1), Marek's Disease Virus (MDV), Pseudorabies Virus (PRV). Special emphasis is put on the exploitation of herpesviral BAC's for the analysis of virus replication und vaccine developments.

Animals↗