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PubMed1994

Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure.

The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped. Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A. vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58. The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1% homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype II strain from wild cherry. The 3' noncoding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 bp fragment derived from the coding sequence of an ipt gene of unknown origin. A comparison of different ipt gene sequences indicates that the corresponding 62 bp sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 bp sequence in the 3' non-coding region. In pTi82.139 the original coding region of the ipt gene has remained largely unmodified. The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 bp repeat in the 3' part of the coding sequence. This leads to the loss of four glutamic acid residues from a series of ten. In spite of these differences, the ipt and 6b genes of pTiAB4 are functional. Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements. Possible implications for the study of bacterial phylogeny are discussed.

Amino Acid Sequence↗

PubMed2003

The spirochetal chpK-chromosomal toxin-antitoxin locus induces growth inhibition of yeast and mycobacteria.

Toxin-antitoxin systems encoded by bacterial plasmids and chromosomes typically consist of a toxin that inhibits growth of the host cell and a specific antitoxin. In this report, the chpK gene from the chromosomal toxin-antitoxin locus of the spirochete Leptospira interrogans was studied in both prokaryotic and eukaryotic systems. Cloning of the the spirochetal chpK gene into a mycobacterial expressing vector led to dramatic reductions of transformation efficiency in both Mycobacterium smegmatis and Mycobacterium bovis BCG. However, few mycobacterial transformants were obtained. This result could be due to plasmid structural modifications leading to disruption of chpK expression, suggesting that L. interrogans ChpK is highly toxic for mycobacteria. Presence of the L. interrogans chpK gene was also found to inhibit cell growth of the yeast Saccharomyces cerevisiae. These results show that ChpK possesses a broad activity against both prokaryotes and eukaryotes, suggesting that the cellular target of the toxin is conserved in these organisms.

Bacterial Toxins↗

PubMed2005

DNA transitions induced by binding of PARP-1 to cruciform structures in supercoiled plasmids.

BACKGROUND: Poly(ADP-ribose)polymerase-1 (PARP-1) binds to single and double-stranded breaks in DNA, but less well known is its affinity for undamaged DNA. Previously, we have shown that PARP-1 also binds to the hairpin structures in DNA models. The mechanism underlying these interactions remains to be defined. METHODS: We analyzed atomic force microscopy (AFM) images of complex of PARP-1 proteins with supercoiled plasmids containing cruciform structures. Using volume measurement analysis of molecules of PARP-1, we determined the numbers of PARP-1 molecules interacting with supercoiled DNA plasmids containing one cruciform structure. We also determined the extent of supercoiling of plasmids. RESULTS: Our observations show that PARP-1 binds to sequences that transition from B-DNA to cruciform structures. PARP-1 is present at the ends of hairpin arms, sites containing a 4-base single-stranded DNA. Furthermore, interaction of PARP-1 with supercoiled plasmids leads to a more relaxed plasmid-DNA conformation. CONCLUSIONS: Binding of PARP-1 to cruciform DNA offers insight into possible mechanisms underlying with changes in DNA conformation. These observations may offer insight into mechanisms involving DNA conformation related to process such as DNA repair and transcription.

Algorithms↗

PubMed1998

Inhibition of ribozymes by deoxyribonucleotides and the origin of DNA.

Two catalytic functions were required, minimally, for the appearance of DNA in evolution: a ribonucleotide reductase (RNR) and a reverse transcriptase (RT). If one accepts the explanatory strength of the RNA world model, it is clear that DNA molecules arose in the RNA world at some stage during the early evolution of cells. I suggest that competition for limited and valuable resources such as nucleotides, amino acids, and sugars made an early appearance among RNA cells, RNA viruses, viroids, and RNA plasmids. Structural and functional similarities between the different types of polymerases favor the simple hypothesis that the first RTs were RNA polymerase mutants that preferentially joined together preexisting deoxyribonucleotide triphosphates (dNTPs) using RNA templates. What was the role of dNTPs inside cells before DNA was synthesized and tested by natural selection? The oxygen atom that is removed by the reductase is of crucial importance to many ribozyme functions, since the 2'-OH is a strong nucleophile that forms transitional states during catalysis. Consequently, a RNR may have been used by cellular parasites to inhibit ribozyme action. Thus, DNA may have been, initially, an inert by-product of retrotranscription in lineages that acquired RTs and could synthesize DNA molecules using cellular RNA templates to detoxify the intracellular environment. DNA was useless as template until a transcriptase (DNA-dependent RNA polymerase) evolved that could copy (-)DNA to reconstitute the (+)RNA genome, indeed a successful way of confronting ribonuclease threats in the RNA world.

DNA↗

PubMed1995

Instability of the mutated biotin operon plasmid in a biotin-producing mutant of Serratia marcescens.

The growth of a d-biotin-producing strain of Serratia marcescens (SB412) was strongly inhibited by the introduction of pLGM412, a low-copy-number plasmid containing the complete biotin (bio) operon derived from SB412, whereas the wild-type strain was not inhibited by the plasmid. SB412 carrying pLGM412 was genetically unstable; large colonies appeared spontaneously from the background small colonies. When the plasmids from the large colonies were transformed into the SB412 host, all of the resultant transformants showed a large-colony phenotype, suggesting that the large-colony phenotype is due to mutations in the plasmid-born bio genes. Some of these plasmids were structurally altered and the others were not. Furthermore, the structurally altered plasmids were classified into a deleted and an elongated type. All of the mutated pLGM412 derivatives reduced or lacked the bio gene expression, indicating that the high expression of bio gene(s) causes the growth inhibition. By subcloning experiments, biotin synthase (the bioB gene product) was responsible for the growth inhibition.

Bacterial Proteins↗

PubMed1997

Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome.

We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this assay, we determined that the AAV Rep proteins, delivered in cis or trans, are required for integration at AAVS1. Only the large forms of the Rep protein, Rep78 and Rep68, promoted site-specific integration. The AAV inverted terminal repeats, present in cis, were not essential for integration at AAVS1, but in cells containing Rep, they increased the efficiency of integration. In the presence of the Rep proteins, the integration of a plasmid containing AAV inverted terminal repeats occurred at high frequency, such that clones containing the plasmid could be isolated without selection. In two of the five clones analyzed by fluorescence in situ hybridization, the plasmid DNA was integrated at AAVS1. In most of the clones, at least one copy of the entire plasmid was integrated in a tandem array. Detailed analysis of the integrated plasmid structure in one clone suggested a complex mechanism producing rearrangements of the flanking genomic DNA, similar to those observed with wild-type AAV.

Adenoviruses, Human↗

PubMed1999

[New knowledge of the molecular biology of enterohemorrhagic Escherichia coli (EHEC) O157].

Since 1982, enterohaemorrhagic Escherichia coli (EHEC) have been identified as a cause of diarrhoea and haemorrhagic colitis. The most serious complication of the infection is the haemolytic-uraemic syndrome (HUS) that develops in 5 to 10% of children with diarrhoea. Shiga toxins (Stx) are the most important presently known virulence factors of EHEC. After reaching the bloodstream, the toxins cause damage of endothelial cells but also of tubular cells in the kidneys which may result in renal failure. In EHEC O157 isolates from patients, we were able to identify seven different combinations of stx genes that occurred with different frequency. The genes encoding Stx are located in the genomes of prophages that are integrated in EHEC chromosomes. In addition, EHEC O157 strains possess a chromosomally located pathogenicity island (pais) termed LEE that contains numerous pathogenicity genes including the eae gene encoding intimin. Moreover, EHEC O157 strains possess a 93-kb plasmid harbouring genes encoding the EHEC-hemolysin (EHEC-HlyA), a serin protease (EspP) that cleaves factor V and a protein called ToxB that shows homology with the toxin B of Clostridium difficile. The EHEC O157 strains exist in two variants, namely non-sorbitol-fermenting (NSF) O157:H7 and sorbitol-fermenting (SF) O157:H- strains that are evolutionary older. Our results obtained up to now demonstrate marked differences in epidemiology of the infection caused by the respective EHEC O157 variants. EHEC O157:H7 strains occur worldwide, whereas SF strains have been hitherto found only in Germany and recently also in the Czech Republic. While the EHEC O157:H7 strains occur mainly during warm months, the SF strains are more frequent during the cold season of the year. In addition, differences exist with regard to the resistance to heavy metals, the plasmid structure, and the reservoir. We postulate that SF O157:H- strains occur only in the human intestine, whereas NSF O157:H7 strains have become adapted to other hosts, such as cattle, promoting a more rapid spread of the strains.

Animals↗

PubMed2001

[Molecular genetic aspects of the antineoplastic effect of gluthoxim].

Gluthoxim--bis(g-L-gluthamyl)-L-cystenyl-bis-glycin disodium salt (C20H32O16N6S2)--is an analog of oxidized gluthation with a stable disulfide bond. It is a drug of a new class of thiopoetins which regulate thiol disulfide metabolism. Gluthoxim is used as a support substance in chemotherapy of cancer. The present investigation deals with feasibility of gluthoxim-induced apoptosis in tumor cell cultures, including human myeloleukema cells HL60 which lack protein p53 gene, so crucial for apoptosis. Also, gluthoxim effect was studied with respect to transformed murine fibroblasts C8 and A4, carrying the plasmide structure with Ras and E1A genes which boost Ras-gene expression. This in turn makes fibroblasts ready to start apoptosis. Gluthoxim induction of cell death was demonstrated in both cell lines; however, apoptosis in cells C8 (with intact gene p53) was much more pronounced than in cells A4 without this gene. It is suggested that apoptosis can be triggered by both imbalance of redox potential typical of tumor cells, induction of p53 synthesis and an impact on the phosphoproteinase cascade of Ras-signal pathway.

Animals↗

PubMed1994

The origins and consequences of genetic instability in prokaryotes.

The causes of genetic change include errors in replication and repair, transposition and recombination. Together, these processes generate a reservoir of genetic variants from which selection will amplify any changes which increase host fitness. Recombination, transposition and slippage during replication may all result in large-scale DNA rearrangements. Homologous and illegitimate recombination generate deletions, inversions, duplications and fusions. Intramolecular transposition can cause deletion or inversion of DNA sequences adjacent to the mobile element, while intermolecular movement may lead to fusion of donor and target replicons when transposition is replicative. Of crucial importance is how genetic variation affects the fitness of the host cell. For a neutral variant the probability of eventual fixation is small, but changes which increase host cell fitness pose a more serious problem because these cells will eventually dominate the culture. The most effective way to combat the proliferation of variant plasmid cloning vectors is to block the processes by which they arise. Cloning vectors should be "stress-tested" and DNA sequence analysis programs can be used to screen for sequence repetition which should be removed, as far as possible, from both vectors and cloned sequences. It is also important that vectors place modest metabolic demands on their hosts in order to minimise the potential increase in fitness associated with changes in plasmid structure.

Biotechnology↗

PubMedDate not supplied

[Formation of cruciform structures in pAO3 plasmid DNA on increasing superhelical density].

The dependence of the crusiciform structure formation on superhelical density was studied by means of high resolution gel-electrophoresis. A short pAO3 DNA plasmid (1683 b. p.) which is a quarter of the ColE1 DNA plasmid and contains the main palindrome of ColE1 DNA was used. The excellent resolution of all topoisomers of pAO3 DNA in gel-electrophoresis made it possible to observe a sharp abruption in the pattern of pAO3 DNA topoisomers separation. The two-dimensional gel-electrophoresis data showed that observed abruption is caused by a sharp decrease of writhing in the molecules with superhelical density--sigma approximately equal to 0,05. An analysis of S1-nuclease digestion products of DNA with different superhelical density was accomplished and these data showed that a sharp structural transition in supercoiled DNA pAO3 is caused by formation of a cruciform structure in the main palindrome.

Base Composition↗

PubMed1991

Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids.

The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.

Base Sequence↗

PubMed1984

P1 plasmid replication: replicon structure.

Bacteriophage P1 lysogenizes Escherichia coli as a unit-copy plasmid. We have undertaken to define the plasmid-encoded elements implicated in P1 plasmid maintenance. We show that a 2081 base-pair fragment of the 90,000 base P1 plasmid confers the capacity for controlled plasmid replication. DNA sequence analysis reveals several open reading frames in this fragment. The largest is shown to encode a 32,000 Mr protein required for plasmid replication. The corresponding gene, repA, has been identified genetically. A set of five 19 base-pair repeats is located upstream from repA; a set of nine similar repeats is located immediately downstream from repA. Each set of repeats, when cloned into pBR322, exerts incompatibility towards a P1 replicon. The upstream set, designated incC, consists of direct repeats that are spaced about two turns of the DNA helix apart; the downstream set, designated incA, consists of nine repeats arranged three in one orientation and six in the other. Spacing between incA repeats were three or four turns of the helix apart. The organization of the plasmid maintenance regions of P1 and the unit-copy sex factor plasmid, F, is strikingly similar. Although the DNA sequences of this region in the two plasmids exhibit little homology, a 9 base-pair sequence that appears four times in the origin region of members of the Enterobacteriaceae also occurs twice as direct repeats in similar positions in P1 and F. This sequence, where it occurs in E. coli, has been postulated to be the binding site for the essential replication protein determined by dnaA. The dnaA protein appears not to be essential for the replication of either plasmid; therefore, the function of the sequence in P1 and F may be regulatory.

Base Sequence↗

PubMed1986

Related structures in the plasmid profiles of Bifidobacterium asteroides, B. indicum and B. globosum.

Seventy strains of B. asteroides isolated from the honey bees Apis mellifera and A. cerana raised in 15 different countries, 73 strains of B. indicum from Apis dorsata and A. cerana from The Philippines and 28 strains of B. globosum isolated from feces of various animals and sewage, were studied for the existence of related structures among their plasmid complements with the Southern blot-hybridization technique. Thirteen different structures were found in B. asteroides and three in B. indicum and B. globosum. A total of twenty five strains were used as source of probes in the hybridization experiments.

Animals↗

PubMed1975

Electron microscopic heteroduplex studies of sequence relations among plasmids of Escherichia coli: structure of F13 and related F-primes.

The structure of F13, a plasmid containing lac, purE, and proC, has been determined by heteroduplex analysis. As expected for an F-prime formed by a type II excision event, it contains all the sequences of F plus a large segment of Escherichia coli chromosomal deoxyribonucleic acid. There is a sequence of F with coordinates 16.3-17.6F which has been shown in other studies to be the insertion sequence IS2. This IS2 occurs twice on F13, once at each of the two junctions of F deoxyribonucleic acid with chromosomal deoxyribonucleic acid. The sequence alpha beta which occurs twice on F with coordinates 93.2-94.5/OF and 13.7-15.0F occurs an additional three times, twice in an inverted order relative to the alpha beta sequences of F, on the chromosomal sequences of F13. The structures of the plasmids F13-4 and F210 have been determined. The common sequences of F13 with F152-1 (a derivative of F152, the classical F2gal) and with F13-4 and F210 have been mapped. These results partially map lac, proC, tsx, and purE on F13. On the basis of all of these results, it is proposed that Hfr 13 (the parent of F13) was formed by recirpocal recombination between IS2 on F and an IS2 resident at a point between lac and proC on the chromosome of the F+ parent of Hfr 13. It is proposed that this IS2 and the several alpha beta sequences on the chromosomal part of F13 are hot spots for recombination with F, i.e., for Hfr formation. The point of origin and direction of transfer of many Hfr's can be explained by this hypothesis. In particular, the sequence relations of F42-1 (Flac) and of F152-1 (F 2gal) with F13 are completely consistent with this model.

Base Sequence↗

PubMed1987

Multicopy plasmid with a structure related to the polyoma virus genome.

In a subclone derived from mouse L(tk-) cells, we found a plasmid present in a high copy number (greater than 5000 copies per cell) that was stably maintained extrachromosomally without any cytopathic effect to the host cells. This plasmid, termed L factor, has two forms: 5.3 and 5.5 kilobase pairs. DNA sequencing and restriction enzyme mapping showed that, although the structure contains DNA sequences common to polyoma virus, plasmid sequences belonging to the regulatory region (the enhancer region) and other regions are quite different from those in polyoma. In cells bearing the plasmid, we detected a low level of material that cross-reacts with antibody to polyoma tumor antigens, suggesting that the plasmids replicate and are maintained in the cells by a mechanism different from that functioning during propagation following infection of papovaviruses.

Animals↗

PubMed1986

Genetic rearrangements of a Rhizobium phaseoli symbiotic plasmid.

Different structural changes of the Sym plasmid were found in a Rhizobium phaseoli strain that loses its symbiotic phenotype at a high frequency. These rearrangements affected both nif genes and Tn5 mob insertions in the plasmid, and in some cases they modified the expression of the bacterium's nodulation ability. One of the rearrangements was more frequent in heat-treated cells, but was also found under standard culture conditions; other structural changes appeared to be related to the conjugal transfer of the plasmid.

DNA Replication↗

PubMed1988

Energy-structure correlations of plasmid DNA in different topological forms.

Differential scanning microcalorimetry (DSC), UV absorption and circular dichroism (CD) have been used to study structure and stability of linear (lin), open circular (oc), supercoiled (cd) and relaxed circular duplex (rd) DNA and calf thymus (CT) DNA. Investigations were made in low salt buffer and in the presence of 7.2 M NaClO4. The chaotropic action of perchlorate promotes a reduction of the overall stability of DNA, which permits a direct determination of the transition enthalpies of all four DNA configurations. The stabilities against thermal denaturation have been found to increase in the series lin approximately oc less than cd less than rd. These relative stabilities can be rationalized on the basis of the linkage between supercoiling and secondary structural changes in topologically constrained duplex DNA. On the basis of these studies, a model of the melting process could be suggested that is consistent with the energetic and spectroscopic data.

Buffers↗

PubMed1994

The organization of the Pm promoter of the TOL plasmid reflects the structure of its cognate activator protein XylS.

The toluate catabolic operon carried by the TOL plasmid pWW0 of Pseudomonas putida is positively regulated by the benzoate-responsive XylS protein which, when activated, stimulates transcription from the operon promoter Pm. In this study, the mode in which XylS effects the activity of the Pm promoter was examined in vivo by genetic analysis of both protein and promoter variants. Substitution of His31Asp/Ser32Pro,Leu113Pro,Phe214Leu/Glu215A sp/Arg216Pro or Thr312Pro, all predicted to disrupt the secondary structure, renders XylS inactive and unable to compete with the native protein for activation of Pm. In contrast, Pro substitutions at Ser64 or Gly160 had no or only minor effects on XylS activity, and a protein with residual capacity to activate Pm resulted when Glu11 was altered to Pro. None of a collection of truncated variants, of XylS protein deleted for more than 10 terminal amino acids retained activity or were able to negatively interact the wild-type XylS protein. These data indicate that amino- and carboxy-terminal portions of the protein sequence depend on each other for the maintenance of their functional structure, rather than being arranged as independent domains. Pm promoter derivatives, in which the two half-sites of the direct repeat sequence that confers responsiveness to XylS were separated and/or inverted by one-half or one complete DNA helix turn, lost responsiveness in vivo. These results favour a model for XylS-mediated activation of Pm in which a tandem dimer is the protein form that binds the Pm promoter.

Amino Acid Sequence↗