Hyperpolymer formation during renaturation of DNA from genomes with different sequence organisation.

Hyperpolymer formation during the renaturation of DNAs from wheat, calf and E. coli was studied using hydroxyapatite chromatography, electron microscopy and S1 nuclease. Large hyperpolymers could not be eluted from hydroxyapatite with 0.5 M phosphate buffer at 60 degrees C. Large proportions of wheat and E. coli DNAs were incorporated into hyperpolymers when fragments 650 nucleotides long were renatured. A much smaller proportion of calf DNA was incorporated under equivalent conditions. Greater proportions of calf DNA accumulated in hyperpolymers only when longer fragments were incubated. Electron microscopy indicated no obvious differences in the basic structures of hyperpolymers formed by the three DNAs and confirmed the quantitative differences in hyperpolymer formation found by hydroxyapatite chromatography. It is concluded that the proportions and arrangement of the repeated sequences in the chromosomes of higher organisms determine the extent of rapid hyperpolymer formation during DNA renaturation in vitro.     

Mitomycin C-induced acceleration of liver cell polyploidization in adult rat after partial hepatectomy

Mitomycin C, a DNA cross-linking agent, was administered for a week intraperitoneally to a normal 9-week-old rat in which hepatocyte proliferative activity had almost ceased. In the rat treated with mitomycin C, the number of polyploid cells with the DNA amounts more than 4C was not increased in comparison with that in the control rat without any treatment. However, the partial hepatectomy in the rat pretreated with mitomycin C provoked prominent hepatocyte polyploidization much greater in degree than that found in the hepatectomized rat without mitomycin C treatment. These results seem to indicate that the existence of cross-linking DNA damages is a latent factor necessary for the induction of abortive mitosis of a cell after completion of DNA synthesis, which results in the production of a mononuclear polyploid cell in one-step higher DNA class. Cross-linking DNA damages and DNA synthesis are the essential factors for the manifestation of polyploidization.     

Kinetics of DNA renaturation catalyzed by the RecA protein of Escherichia coli

The recA enzyme of Escherichia coli catalyzes renaturation of DNA coupled to hydrolysis of ATP. The rate of enzymatic renaturation is linearly dependent on recA protein concentration and shows saturation kinetics with respect to DNA concentration. The kinetic analysis of the reaction indicates that the Km for DNA is 65 microM while the kcat is approximately 48 pmol of duplex formed (pmol of recA)-1 (20 min)-1. RecA protein catalyzed renaturation has been characterized with respect to salt sensitivity, Mg2+ ion and pH optima, requirements for nucleoside triphosphates, and inhibition by nonhydrolyzable nucleoside triphosphates and analogues. These results are consistent with a Michaelis-Menten mechanism for DNA renaturation catalyzed by recA protein. A model is described in which oligomers of recA protein bind rapidly to single-stranded DNA, and in the presence of ATP, these nucleoprotein intermediates aggregate to bring complementary sequences into close proximity for homologous pairing. As with other DNA pairing reactions catalyzed by recA protein, ongoing DNA hydrolysis is required for renaturation. However, unlike the strand assimilation or transfer reaction, renaturation is inhibited by E. coli helix-destabilizing protein.     

Renaturation effects of cis and trans platinum II and IV compounds on calf thymus deoxyribonucleic acid.

The effects of cis dichlorodiammine platinum [cis Pt(II)], trans dichlorodiammine platinum (trans Pt(II)], cis tetrachlorodiammine platinum [cis Pt(IV)], trans tetrachlorodiammine platinum [trans Pt(IV)], and ethylenediaminedichloride platinum [Pt(II)en] on the absorption spectra, and thermal hyper- and hypochromicity of calf thymus DNA were investigated. Platinum-induced renaturation was studied as one parameter of interstrand cross-linking. Based on a DNA cross-linking hypothesis, the tumor-inhibitory platinum compounds cis Pt(II), cis Pt(IV) and Pt(II)en would be expected to induce renaturation following thermal denaturation, whereas the ineffective drugs, trans Pt(II) and trans Pt(IV) would not. All five bind to DNA in such a way as to induce renaturation. However, cis Pt(IV) requires at least a 3- to 4-fold longer incubation time than is required by the other compounds to form the coordination bonds necessary for renaturation. Maximum renaturation with all compounds was observed at a molar Pt/base ratio of 0.05 except cis Pt(IV), with which it was 0.25. The rate of the formation of the platinum-coordinated cross-links by fresh cis Pt(II) suggests two reactions or types of reactions occur. The first is rapid and destabilizes the DNA helix, whereas the second is slow and responsible for renaturation following thermal denaturation. These results suggest that cis Pt(IV) may be activated cellularly and that cross-linking is not the primary mechanism of action of the tumor-inhibitory platinum compounds.     

Protective activity of the antilipopolysaccharide antibodies from human cord serum

We evaluated the ability of human maternal and cord serum antibodies to protect mice challenged with live Escherichia coli serotype O6:K2ac (E. coli O6). Mice received paired maternal or cord serum pools before a challenge with E. coli O6 to evaluate the mortality rate. All the pools were able to protect the animals challenged with bacteria except the test group from paired maternal and cord sera from preterm neonates containing less than 1.0 mg L(-1) immunoglobulin G antibody levels. In liver, spleen and mesenteric lymph nodes from the control group (phosphate-buffered saline), more than 10(2) CFU mL(-1) bacteria were found at 30 min and more than 10(5) CFU mL(-1) after 120 min. The test group showed lower bacterial counts in the organs, and no bacteria in the mesenteric lymph nodes during the evaluated period. Tumor necrosis factor alpha and interleukin 6 were undetectable in serum from animals pretreated with paired maternal and cord serum pools from full-term neonates and pools from preterm neonates containing high antibody and avidity levels. Our findings suggest that placental transfer of antilipopolysaccharide O6 immunoglobulin G antibodies to neonates has a high capacity to prevent lethal infection with E. coli O6 in a mouse protection model and that the degree of protection is determined by the concentration and avidity of these IgG antibodies.     

Influence of antibodies against DNA on the renaturation of DNA.

The treatment of denatured T4 phage DNA with antiserum for the DNA of this phage, containing antibodies against glucosylated 5-hydroxymethylcytosine, decreases the ability of DNA for renaturation. The greatest inhibiting activity is possessed by antiserum for T4 phage DNA irradiated with UV light, which contains antibodies not only against glucosylated 5-hydroxymethylcytosine, but also against the usual nitrogen bases. Antiserum against E. coli DNA, containing antibodies to the usual nitrogen bases, in equal dilutions with the antisera indicated above, shows less inhibitory activity on the renaturation of T4 phage DNA.     

Temperature dependence of the photosynthetic activities in the thylakoid membranes from the blue-green alga Anacystis nidulans.

The effects of temperature and ethidium bromide on the banding of heat-denatured DNA was studied during equilibrium centrifugation in density gradients of NaI. Centrifugation at 10 degrees C prevents the partial renaturation of Escherichia coli DNA and Clostridium perfringens DNA that occurs at 20 degrees C. A centrifugation temperature of --5 degrees C is required to prevent renaturation of T7 phage DNA. Ethidium bromide decreases renaturation of Escherichia coli DNA during centrifugation at 20 degrees C and causes a small shift in the buoyant density of both denatured and native DNA. Equilibrium centrifugation at lower temperatures prevents DNA renaturation and permits increased utilization of the large buoyant density difference between native and heat-denatured DNA in gradients of NaI.     

The interaction of dianhydrogalactitol with DNA in cultured Yoshida sarcoma cells

Using alkaline sucrose gradient sedimentation centrifugation it was found that treatment of Yoshida sarcoma cells in culture for 1 h with increasing concentrations of dianhydrogalactitol (DAG) enhanced the sedimentation rate of DNA in a dose-dependent manner. There was no difference between the amount of protein which co-sedimented with DNA released from treated and untreated cells. When DNA was extracted from the cells using a p-amino-salicylate-phenol mixture, the protein content of DNA seemed not to be affected by DAG. The possibility that DAG could form interstrand cross-linking in cellular DNA was suggested from renaturation studies. The appearance of a fast sedimenting DNA in the alkaline sucrose gradient and the evidence for a cross-linked DNA detected by renaturation technique, only appeared later than 6 h after treatment. A similar delayed effect on the depression in the rate of DNA synthesis was also observed. These data suggest that the inhibition of DNA synthesis may be related to the delayed formation of DNA interstrand cross-linked.     

Rapidly labelled ribonucleic acid from rat liver. Studies in the attachment to ribosomes and stimulation of the incorporation of amino acids into polypeptides in cell-free systems

1. Rapidly labelled RNA from rat liver, either as a complex with DNA (m-RNA-DNA) or with ribosomal RNA (m-RNA-RNA) binds to ribosomes in the polysome region. No binding could be demonstrated with ribosomal RNA or native DNA from Bacillus subtilis. 2. With ribosomes from rat liver, Escherichia coli or hepatoma the m-RNA-DNA stimulated incorporation of amino acids with rat-liver ribosomes only, whereas the m-RNA-RNA complex was effective with ribosomes from E. coli or the hepatoma. 3. Polyuridylic acid was effective as messenger RNA with all three ribosomes but much greater stimulation was obtained with ribosomes from E. coli and the hepatoma. 4. The degree of incorporation of phenylalanine with polyuridylic acid and ribosomes from a hepatoma was decreased by about 50% when ribosomal RNA was present.     

Characterization of a murine model of monocrotaline pyrrole-induced acute lung injury

Background

New animal models of chronic pulmonary hypertension in mice are needed. The injection of monocrotaline is an established model of pulmonary hypertension in rats. The aim of this study was to establish a murine model of pulmonary hypertension by injection of the active metabolite, monocrotaline pyrrole.

Methods

Survival studies, computed tomographic scanning, histology, bronchoalveolar lavage were performed, and arterial blood gases and hemodynamics were measured in animals which received an intravenous injection of different doses of monocrotaline pyrrole.

Results

Monocrotaline pyrrole induced pulmonary hypertension in Sprague Dawley rats. When injected into mice, monocrotaline pyrrole induced dose-dependant mortality in C57Bl6/N and BALB/c mice (dose range 6–15 mg/kg bodyweight). At a dose of 10 mg/kg bodyweight, mice developed a typical early-phase acute lung injury, characterized by lung edema, neutrophil influx, hypoxemia and reduced lung compliance. In the late phase, monocrotaline pyrrole injection resulted in limited lung fibrosis and no obvious pulmonary hypertension.

Conclusion

Monocrotaline and monocrotaline pyrrole pneumotoxicity substantially differs between the animal species.     

Evaluation of the heat inactivation of Escherichia coli and Lactobacillus plantarum by differential scanning calorimetry

Differential scanning calorimetry (DSC) is used to evaluate the thermal stability and reversibility after heat treatment of transitions associated with various cellular components of Escherichia coli and Lactobacillus plantarum. The reversibility and the change in the thermal stability of individual transitions are evaluated by a second temperature scan after preheating in the DSC to various temperatures between 40 and 130 degrees C. The viability of bacteria after a heat treatment between 55 and 70 degrees C in the DSC is determined by both plate count and calorimetric data. The fractional viability values based on calorimetric and plate count data show a linear relationship. Viability loss and the irreversible change in DSC thermograms of pretreated whole cells are highly correlated between 55 and 70 degrees C. Comparison of DSC scans for isolated ribosomes shows that the thermal stability of E. coli ribosomes is greater than that of L. plantarum ribosomes, consistent with the greater thermal tolerance of E. coli observed from viability loss and DSC scans of whole cells.     

Protein targets of monocrotaline pyrrole in pulmonary artery endothelial cells

A single administration of monocrotaline to rats results in pathologic alterations in the lung and heart similar to human pulmonary hypertension. In order to produce these lesions, monocrotaline is oxidized to monocrotaline pyrrole in the liver followed by hematogenous transport to the lung where it injures pulmonary endothelium. In this study, we determined specific endothelial targets for (14)C-monocrotaline pyrrole using two-dimensional gel electrophoresis and autoradiographic detection of protein metabolite adducts. Selective labeling of specific proteins was observed. Labeled proteins were digested with trypsin, and the resulting peptides were analyzed using matrix-assisted laser desorption ionization mass spectrometry. The results were searched against sequence data bases to identify the adducted proteins. Five abundant adducted proteins were identified as galectin-1, protein-disulfide isomerase, probable protein-disulfide isomerase (ER60), beta- or gamma-cytoplasmic actin, and cytoskeletal tropomyosin (TM30-NM). With the exception of actin, the proteins identified in this study have never been identified as potential targets for pyrroles, and the majority of these proteins have either received no or minimal attention as targets for other electrophilic compounds. The known functions of these proteins are discussed in terms of their potential for explaining the pulmonary toxicity of monocrotaline.     

Refolding and structural characterization of the human p53 tumor suppressor protein

The human tumor suppressor p53 is a conformationally flexible and functionally complex protein that is only partially understood on a structural level. We expressed full-length p53 in the cytosol of Escherichia coli as inclusion bodies. To obtain active, recombinant p53, we varied renaturation conditions using DNA binding activity and oligomeric state as criteria for successful refolding. The optimized renaturation protocol allows the refolding of active, DNA binding p53 with correct quaternary structure and domain contact interfaces. The purified protein could be allosterically activated for DNA binding by addition of a C-terminally binding antibody. Analytical gelfiltration and chemical cross-linking confirmed the tetrameric quaternary structure and the spectroscopic analysis of renatured p53 by fluorescence and circular dichroism, suggested that native p53 is partially unstructured.     

Kinetic and spectrophotometric studies on the renaturation of deoxyribonucleic acid

The kinetics of the renaturation of Escherichia coli DNA in 0.4-1.0m-sodium chloride at temperatures from 60 degrees to 90 degrees have been studied. The extent of renaturation was a maximum at 65 degrees to 75 degrees and increased with ionic strength, and the rate constant increased with both ionic strength and temperature. The energy and entropy of activation of renaturation were calculated to be 6-7kcal.mole(-1) and -40cal.deg.(-1)mole(-1) respectively. It has been shown that renaturation is a second-order process for 5hr. under most conditions. The results are consistent with a reaction in which the rate-controlling step is the diffusion together of two separated complementary DNA strands and the formation of a nucleus of base pairs between them. The kinetics of the renaturation of T7-phage DNA and Bordetella pertussis DNA have also been studied, and their rates of renaturation related quantitatively to the relative heterogeneity of the DNA samples. By analysis of the spectra of DNA at different stages during renaturation it was shown that initially the renatured DNA was rich in guanine-cytosine base pairs and non-random in base sequence, but that, as equilibrium was approached, the renatured DNA gradually resembled native DNA more closely. The rate constant for the renaturation of guanine-cytosine base pairs was slightly higher than for adenine-thymine base pairs.     

Escherichia coli single-stranded DNA binding protein stimulates the DNA deoxyribophosphodiesterase activity of exonuclease I.

The E. coli single-stranded binding protein (SSB) has been demonstrated in vitro to be involved in a number of replicative, DNA renaturation, and protective functions. It was shown previously that SSB can interact with exonuclease I to stimulate the hydrolysis of single-stranded DNA. We demonstrate here that E. coli SSB can also enhance the DNA deoxyribophosphodiesterase (dRpase) activity of exonuclease I by stimulating the release of 2-deoxyribose-5-phosphate from a DNA substrate containing AP endonuclease-incised AP sites, and the release of 4-hydroxy-2-pentenal-5-phosphate from a DNA substrate containing AP lyase-incised AP sites. E. coli SSB and exonuclease I form a protein complex as demonstrated by Superose 12 gel filtration chromatography. These results suggest that SSB may have an important role in the DNA base excision repair pathway.     

Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli.

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Thermostability of DNA and its connection with the glassing process

Using differential scanning calorimetry, the thermal denaturation of calf thymus DNA with different content of water (from 12 to 92%) was investigated. Dependences of melting temperature and enthalpy on the biopolymer hydration degree were established. Within the range of water concentrations from 92 to 50% the values of thermodynamic parameters of denaturation were obtained being in good agreement with the published data. Besides, a calorimetric manifestation of renaturation process at different cooling conditions after denaturation was studied. Special attention was paid to thermal properties of denatured and native DNA in the samples containing only the bound water. The temperature dependence of heat capacity in the denatured samples, which have completely lost their renaturation ability due to the proper thermal treatment, demonstrated a characteristic jump of thermal capacity. The value of this jump has been determined to be equal to 1.0 cal/g. degree C, related to dry weight, and almost not dependent on humidity. Temperature position of the jump (Tg) depends on the content of water which serves as a plasticizer. It is shown that the observed anomaly demonstrates all the properties characteristic of vitrification process in synthetic polymers and proteins. General similarity of thermal properties of the samples of native DNA, containing only the bound water, with those of denatured DNA also indicates a transition from the glassy into the rabber-like state. A possibility of existence of both native and denatured DNA in the glassy state at room temperature for the samples with low humidity (about 25%) has been demonstrated experimentally. It can be suggested that the formation of glassy state at dehydration of native DNA ensures its thermostability and the ability of restoration of its functional properties at a subsequent dehydration.     

Sequence analysis of the Legionella micdadei groELS operon

A 2.7 kb DNA fragment encoding the 60 kDa common antigen (CA) and a 13 kDa protein of Legionella micdadei was sequenced. Two open reading frames of 57,677 and 10,456 Da were identified, corresponding to the heat shock proteins GroEL and GroES, respectively. Typical -35, -10, and Shine-Dalgarno heat shock expression signals were identified upstream of the L. micdadei groEL gene. Further upstream, a poly-T region, also a feature of the sigma 32-regulated Escherichia coli groELS heat shock operon, was found. Despite the high degree of homology of the expression signals in E. coli and L. micdadei, Western blot analysis with an L. micdadei specific anti-groEL antibody did not reveal a significant increase in the amount of the GroEL protein during heat shock in L. micdadei or in the recombinant E. coli expressing L. micdadei GroEL.     

Separation of genomic DNA from plasmid DNA by selective renaturation with immobilized metal affinity capture

In contrast to proteins, many nucleic acids can undergo reversible modification of their conformations, and this flexibility can be used to facilitate purification. Selective renaturation with capture is a novel method of removing contaminating genomic DNA from plasmid samples. Plasmid DNA quickly renatures after thermal denaturation and cooling (or alkaline denaturation followed by neutralization), whereas genomic DNA remains locally denatured after rapid cooling in mismatch-stabilizing high ionic strength buffer. Partially denatured genomic DNA can be selectively bound to a metal chelate affinity adsorbent through exposed purine bases, while double-stranded renatured plasmid DNA is not bound. Using this method we have readily achieved 1,000,000-fold clearance of 71 wt % contaminating E. coli genomic DNA from plasmid samples.     

A grpE mutant of Escherichia coli is more resistant to heat than the wild-type

The grpE gene of Escherichia coli is essential for bacteriophage lambda DNA replication and is also necessary for host RNA and DNA synthesis at high temperature. A grpE mutant of E. coli was found to be substantially more resistant to 50 degrees C heat treatment than the wild-type. Upon receiving a 42 degrees C heat shock for 15 min, both the wild-type and the grpE mutant became more resistant to heat (i.e. they became thermotolerant). A grpE+ revertant behaved similarly to the wild-type in that it was more sensitive to heat than grpE cells. In addition, grpE cells had the same H2O2 and UV sensitivity as the wild-type. This implies that the conditions for which a grpE mutation is beneficial are unique to heat exposure and are not caused by H2O2 or UV exposure. Furthermore, synthesis of heat-shock proteins occurred sooner in the grpE mutant than in the wild-type, indicating that the grpE gene of E. coli may influence the regulation of the heat-shock response.