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1.
M. Thomas Record 《Biopolymers》1975,14(10):2137-2158
The effects of monovalent (Na+) and divalent (Mg++) cations on the temperature and breadth of the helix–coil transition of phage DNA have been investigated. The experimental results confirm the findings of Dove and Davidson [J. Mol. Biol. 5 , 467–478 (1962)] for the limiting cases of zero divalent ion concentration and saturating levels of divalent ion, and extend their findings to the intermediate region of Mg++ concentrations. A theory for the dependence of transition temperature on the ion concentrations is developed, utilizing the approach of Wyman [Adv. Protein Chem. 19 , 223–286 (1964)], modified to account for electrostatic nonideality of the polyelectrolytes. The theory is in agreement with Manning's treatment of the experiments of Dove and Davidson [Biopolymers 11 , 937–949, 951–955 (1972)] and is in fair agreement with experimental data over the entire range of ion concentrations. Further investigation of the structure and ion-binding properties of the denatured form will be required before a quantitative comparison between theory and experiment can be performed.  相似文献   

2.
Kinetics of the helix-coil transition in DNA   总被引:2,自引:0,他引:2  
M T Record 《Biopolymers》1972,11(7):1435-1484
The kinetics of the helix-coil transition have been investigated for T2 and T7 phage DNA in a formamide-water-salt mixed solvent using a slow temperature perturbation technique (applicable to kinetic processes with rate constants ? 3 min?1). In this solvent degradation of the DNA is effectively suppressed. Complex kinetic curves are observed by absorbance and viscosity measurements for the response to denaturing perturbations in the transition region. Analysis of the decay curves indicates that the denaturation reaction in this time range can be treated as a first-order reaction with a variable first-order rate parameter, k, the derivative of the logarithm of the absorbance or viscosity change with respect to time. In the approach to denaturation equilibrium in the transition region, the rate parameter is determined only by the instantaneous extent of denaturation of the molecules. Near equilibrium, the rate parameter assumes a constant value characteristic of the equilibrium state. In this region, where the denaturation reaction proceeds as a simple first-order process, both the decay of absorbance (reflected local conformational change) and the decay of solution viscosity (reflecting macromolecular conformational change) are characterized by the same constant value of k. In 83% formamide, 0.3M Na+, the rate parameter k for T2 DNA decreases from an extrapolated value of 2.0 min?1 at 0% denaturation to 0.11 min?1 at 90% denaturation. Rate parameters determined for T7 DNA at the same counterion concentration and fraction of denaturation are approximately five times as large as those cited for T2 DNA, indicating an inverse proportionality of rate constant to molecular length. On the other hand, simple first-order kinetic responses with constant k are obtained for renaturing perturbations within the transition, indicating that the mechanism of rewinding differs, in most cases, from that of unwinding. Only in the limit of very small perturbations about a given equilibrium position are the rate constants k obtained from denaturing and renaturing perturbations equal. For perturbations of finite size, it appears possible that an intramolecular initiation or nucleation event may precede rewinding and limit the rate of this reaction. The rate parameters again are approximately inversely proportional to molecular weight. The one exception to the first-power dependence on molecular weight appears when temperature jumps are made upward into the post-transition region. Here the molecular-weight dependence is second power, but complications arising from the different strand-separation properties of T2 and T7 DNA's make interpretation difficult. The previously used model of friction-limited unwinding appears to fit all the observations except for the molecular-weight dependence.  相似文献   

3.
Six bacteriophages with an elongated head and a short, noncontractile tail were compared by DNA-DNA hybridization, seroneutralization kinetics, mol% G+C and molecular weight of DNA, and host range. Three phage species could be identified. Phage species 1 containedEnterobacter sakazakii phage C2,Erwinia herbicola phages E3 and E16P, andSalmonella newport phage 7–11. These phages had a rather wide host range (4 to 13 bacterial species). DNA relatedness among species 1 phages was above 75% relative binding ratio (S1 nuclease method, 60°C) when labeled DNA from phage C2 was used, and above 41% when labeled DNA from phage E3 was used. Molecular weight of DNA was about 58×106 (C2) to 67 ×106 (E3). The mol% G+C of DNA was 43–45. Anti-C2 serum that neutralizes all phages of species 1 does not neutralize phages of the other two species. Species 2 contains only coliphage Esc-7-11, whose host range was only oneEscherichia coli strain out of 188 strains of Enterobacteriaceae studied; it was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage Esc-7-11 had a base composition of 43 mol% G+C and a molecular weight of about 45×106. Species 3 contains onlyProteus mirabilis phage 13/3a. Its host range was limited to swarmingProteus species. Species 3 was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage 13/3a had a base composition of 35 mol% G+C and molecular weight of about 53×106. It is proposed that phage species be defined as phage nucleic acid hybridization groups.  相似文献   

4.
A new virulent bacteriophage, designated RZ1, was isolated from a local pond on the facultative phototrophic bacteriumRhodobacter sphaeroides ZZ101. Electron microscopic studies revealed that, in general morphology, phage RZ1 resembles the bacteriophage ofEscherichia coli. The host range of phage RZ1 is limited to some strains ofR. sphaeroides. The phage genome consists of double-stranded DNA of about 44 kb lacking cohesive ends and seems to present terminal redundancy and cyclic permutation. RZ1 phage may carry out a lytic cycle only in recombination-defective mutants ofR. sphaeroides. Nevertheless, a derivative of the RZ1 phage, termed RW1, able to grow in recombination-proficient strains ofR. sphaeroides, has also been obtained. In vitro restriction analysis of both RZ1 and RW1 phages shows the presence of a rearrangement in their DNA. Generalized transduction of Strr and Rifr chromosomal markers has not been detected with either RZ1 or RW1 phages.  相似文献   

5.
Interactions of meso-tetra(4-N-methylpyridyl)porphyrin [TMpyP(4)], meso-tetra(2-N-methylpyridyl)porphyrin [TMpyP(2)], and meso-tetra(para-N-trimethylanilinium)porphyrin (TMAP) with several native and synthetic DNAs were studied by a variety of physical techniques: nmr (31P and 1H), absorption spectroscopy, viscosity, and flow dichroism (FD). Of the three porphyrins studied, only the interaction of TMpyP(4) with poly [d(G-C)2] was fully consistent with intercalation. In particular, a large increase in viscosity, a downfield 31P-nmr signal (ca. -1 ppm), and upfield imino proton signals (11 to 12 ppm range) were observed. Comparison of the effects of TMpyP(4) on DNAs of different GC contents revealed larger changes in solution viscosity with increased GC content. However, the characteristic changes in 31P- and 1H-nmr spectra were not observed. The viscosity increases observed in studies with poly[d(A-C)(G-T)] and C. Perf. DNA were much lower than with poly[d(G-C)2], M. Lys. DNA, and calf thymus DNA. Thus, GC sequence and content are clearly important. The principal change in the 31P-nmr signal of native DNA is the appearance of a very broad shoulder centered at ca. -2.0 ppm, which is larger in M. Lys. DNA than in C. Perf. DNA. FD studies indicate highly ordered TMpyP(4) cations arranged perpendicular to the DNA axis of calf thymus DNA. Together, these results suggest the major effects of TMpyP(4) on DNA properties are due to strong GC-binding interactions that influence DNA structure. The data are consistent with combined intercalative and outside binding interactions of TMpyP(4) with GC regions of DNA. In contrast, similar studies with TMAP suggest that it influences AT regions of DNA by an outside binding mode. On the other hand, TMpyP(2) effects on DNA properties are consistent with nonselective outside binding.  相似文献   

6.
Summary The following properties of the DNA of B. subtilis phage SP50 were established: Molecular weight (in Daltons) 102×106 (sedimentation velocity) 97×106 (viscosity) 97×106 (contour lengths of electron micrographs) Base Composition (in % GC) 41.7 (chemical analysis) 44 (melting point) 44 (buoyant density) No unusual bases were observed. The complementary strands of the DNA can be separated. The phage DNA has genuine single strand breaks. The number and distribution of such breaks appears to be determined by the host on which phages were grown.This investigation was supported in part by a Public Health Service research grant GM 13,666 from the National Institutes of General Medical Sciences, AI 01267 from the National Institutes of Allergy and Infectious Diseases, AM 04763 from the National Institutes of Arthritis and Metabolic Diseases; cancer research funds from the University of California; and a grant from the Hartford Foundation.  相似文献   

7.
The natural transformation of Acinetobacter calcoaceticus BD413 (trp E27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp +) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca2+. With CaCl2 solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl2 and MgCl2. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80°C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.  相似文献   

8.
Sd phage were incubated in 1 m-O-methylhydroxylamine. At various time-intervals, samples of modified phage were isolated and disrupted either by heating or by treatment with detergent. Changes in viscosity and buoyant density of disrupted preparations took place in the course of modification. Three transient synchronous drops in viscosity and buoyant density levels were observed with minima at five minutes, one and three hours of modification. The specific viscosity of the preparations at minima was 10 to 20% that of the disrupted unmodified phage.Properties of the phage preparation isolated during the third period of decreased viscosity were studied in more detail. This preparation, subjected to thermal disruption, gives a single DNA-containing band in Cs2SO4 gradient centrifugation corresponding to a buoyant density of 1.37 g/cm3 (cf. 1.39, 1.29 and 1.43 g/cm3 for whole phage, phage ghosts and native phage DNA, respectively).The band contains practically all the 35S label that was present in the starting phage, suggesting that it corresponds to a complex of phage DNA with protein. Electron microscopy revealed complexes as thick strands of 50 to 300 Å diameter bonded to globular particles of varying size.In four hours of modification, the viscosity and buoyant density of disrupted phage returned to values characteristic of unmodified preparations. The DNA band contained no 35S label. Electron microscopy of the substance of this band revealed fibres of 20 Å diameter.A possible explanation of the results is based on the assumption of pre-existing non-covalent interaction of C(4)—NH2 moieties of cytidine residues with nucleophilic groupings of coating protein within the virion. It is assumed that it is this interaction that holds DNA in “non-native” conformation within intact phage particles and thus explains its peculiar properties discovered earlier. In the present case, the interaction determines the formation of DNA-protein crosslinks under O-methylhydroxylamine treatment via the earlier postulated intermediate product of cytosine modification. Restoration of “normal” physical properties of disrupted phage after more prolonged modification is explained by cleavage of the DNA-protein cross-links due to reaction of the postulated intermediate with O-methylhydroxylamine affording N(4)-methoxy-6-methoxy-amino-5,6-dihydrocytidine residues.  相似文献   

9.
Summary The UV-sensitivity of phage and its infectious DNA have been compared in experiments involving infection of normal cells by phage and transfection of lysozyme-EDTA spheroplasts or Ca++-treated cells by phage DNA. It is shown that UV-irradiated DNA undergoes extensive HCR. Since intact phage and free phage DNA have the same survival after UV-irradiation in Hcr- spheroplasts and cells, resp., and since survival is also identical in Ca++-treated Hcr+ cells it is concluded that DNA in solution or packaged in the phage head provides the same target for the induction of lethal UV lesions. This conclusion is supported by the observation that cysteamine provides a similar radioprotection to the intact phage and its free DNA. Spheroplasts of Hcr+ cells, however, have an HCR capacity reduced by about 20% when compared with normal or Ca++-treated cells. Moreover, UV-reactivation of irradiated DNA, which is absent in spheroplasts, occurs efficiently in Ca++-treated cells. Possible reasons for the physiological difference between spheroplasts and normal cells are discussed. c-mutations, which are readily induced by UV in phage assayed with E. coli mul -, could not be induced in DNA when assayed with spheroplasts or Ca++-treated cells of this strain. No mutants were also found with DNA extracted from UV-irradiated phage. The significance of the mode of entry of UV-irradiated DNA into a cell for the production of mutations is discussed.  相似文献   

10.
Summary We demonstrate the use of bacteriophage P4 as a molecular cloning vector in Klebsiella pneumoniae. A hybrid P4 phage, constructed in vitro, that contains a K. pneumoniae hisDG DNA fragment can be propagated either as a lytic viable specialized transducing phage or as an autonomous, self-replicating plasmid. Hybrid P4 genomes existing as plasmids can be readily converted into non-defective P4-hybrid phage particles by superinfection with helper phage P2. Infection of a K. pneumoniae hisD non-P2 lysogen with P4-hisD hybrid phage results in approximately 90% of the infected cells becoming stably transduced to HisD+. Because P4 interferes with P2 growth, high titre stocks of P4 hybrid phages are relatively free (10-6) of P2 contamination. The hisG gene product was detected in ultraviolet light irradiated host cells infected by the P4-hisDG hybrid phage. A mutant of P4 (P4sidl) that directs the packaging of P4 DNA into P2 sized capsids should permit the construction of hybrid phages carrying 26 kilobase inserts.  相似文献   

11.
Summary The EcoRI digestion products of phage T4 DNA have been examined using a phage DNA transformation assay. A 2.6x106 Dalton fragment was found to contain the rII genes. This fragment was purified and then treated with HindIII endonuclease. The cleavage products were ligated to the vector plasmid pBR313 and viable recombinant plasmids recovered. A genetic assay was employed to demonstrate that the recombinants contained T4 DNA and to localize on the phage genetic map the EcoRI and HindIII sites cleaved during the construction of the plasmids. Preliminary characterization suggests that a fragment covering the beginning of the rIIA gene possibly contains a promotor which is active in uninfected cells.Abbreviations used Ap ampicillin - Tc tetracycline - Mdal 106 Daltons - bp base pairs  相似文献   

12.
O Gotoh  A Wada  S Yabuki 《Biopolymers》1979,18(4):805-824
Melting profiles of DNAs from wild-type λ phage and a deletion mutant phage λb2 were examined in a wide range of salt concentration. The fine structure of the melting profiles changed sharply with salt concentration, especially in the range [Na+] ? 10 mM. A comparison of the melting profiles between the wild-type and the deletion mutant DNAs provided good evidence for extremely high melting cooperativity under low salt conditions, which is clearly manifested as the long-range interactions and the pronounced end effects; a large melting peak appeared as a result of the b2 deletion without any inserted sequence in the salt range [Na+] ? 2.8 mM. It was also suggested that in the further reduced salt range [Na+] ? 2.0 mM, melting of a λ DNA molecule starts from its right end rather than the most (A + T)-rich central region. The molecular basis of the high melting cooperativity at low salt concentrations can be explained in terms of the increased free energy associated with loop formation in the double-helical structure of DNA.  相似文献   

13.
Summary We have detected in vitro homologous recombination mediated by purified recA protein of Escherichia coli as a recombinant phage produced by using the DNA packaging system of phage . When double-stranded DNA of phage carrying amber mutations is incubated with double-stranded DNA carrying the wild-type genes in the presence of recA protein, Mg++ and ATP, and the DNA packaged, amber + recombinant phage is produced at a high frequency. This reaction depends completely upon the function of the wild-type recA protein. After incubation of 32P-labeled linear DNA (Form III) with bromouracil-labeled circular DNA (Form I-Form II mixture) in the presence of recA protein, Mg++ and ATP, about 10% of the 32P-counts band at an intermediate density in CsCl equilibrium gradient. This fraction yields a high percentage of the recombinant phage after DNA packaging and shows the -shaped and -shaped joint molecules of linear and circular DNA under the electron microscope. Furthermore, we demonstrate that a non-homologous region inhibits the recombination reaction when it is between the marker concerned and the closer cos end. Our results indicate thatrecA protein acts directly in the initial step of recombination to join the homologous double-stranded DNA and that the resulting molecule can be matured into the recombinant DNA.Abbreviations kb kilobase pairs - PFU plaque forming units - Form I superhelical closed circular DNA - Form II open circular DNA - Form III linear DNA  相似文献   

14.
H P Hopkins  W D Wilson 《Biopolymers》1987,26(8):1347-1355
Enthalpy changes (ΔHB) for the binding of ethidium (a monocation) and propidium (a dication) to calf thymus DNA have been determined calorimetrically in piperazine-N, N′-bis(2-ethanesulfonic acid) buffer with the fluoride ion as the counterion. Heats of dilution for the fluoride salts of ethidium and propidium were substantially less than the corresponding values found for other halide salts of these cations. At a Na+ ion concentrations of 0.019, ΔHB = ?8.3 and ?7.9 ± 0.3 kcal mol?1 for ethidium and propidium, respectively. For these two cations, just as was observed for the naphthalene monoimide (monocation) and diimide (dication) [H. P. Hopkins, K. A. Stevenson, and W. D. Wilson, (1986) J. Sol. Chem. 15 , 563–579], ΔHB is within the same experimental error for both cations. Apparently, charge–charge interactions in DNA–cation complexes produce only small changes in the enthalpy for the system. In the concentration range 0.019–0.207, the ΔHB values for propidium did not depend appreciably on the Na+ ion concentration, and a similar pattern was shown to exist for ethidium. When these results were combined with ΔGB values for the binding of these cations to DNA, we found the variation of ΔSB with Na+ ion concentration to be remarkably close to the predictions of modern polyelectrolyte theory, i.e., propidium binding to DNA causes approximately twice as many Na+ ions to be released into the bulk solution as does the binding of ethidium. The much stronger binding of propidium, relative to ethidium, at low ionic strengths is thus seen to be primarily due to entropic effects.  相似文献   

15.
Summary Tn7 insertions into the genome of F116L, a Pseudomonas aeruginosa generalized transducing phage, were isolated by repeated cycles of transducing phage, were of strains lysogenic for F116cts mutants with selection for trimethoprim resistance (Tp1). Two non-defective F116Lcts:Tn7 phage were characterized. They have reduced plaquing ability, produced non-lysogenic Tpr transductants, and have yielded a deletion mutant of the phage genome upon selection for plaque formation in single infection. F116L DNA is circularly permuted and terminally redundant. A circular restriction map of 61.7 kb has been defined, and a cleavage site common to many enzymes has been identified at coordinate 23.3 kb on the map. It is presumed that this site represents the sequence for the initiation of DNA encapsidation by a headful packaging mode. The Tn7 insertion targets and a 13.4 kb deletion define regions of the F116L genome non-essential for either vegetative growth or lysogenization. The restriction map of Tn7 has been determined for five enzymes. Non-lysogenic Tpr transuctants reveal a Tn7 insertion hot-spot in the P. aeruginosa genome.  相似文献   

16.
Summary The DNA of a gene 2 mutant (T4 2 ) of phage T4 is degraded by RecBCD enzyme in the bacterial cytoplasm. Under normal conditions, recBCD + cells are therefore incapable of supporting the growth of phage T4 2 . Only if the nucleolytic activity of RecBCD enzyme is absent from the cytoplasm are T4 2 -infected bacteria able to form plaques. We found that recBCD + cells can form plaques if, before infection with T4 2 , they have been exposed to gamma radiation. It is suggested that gamma ray-induced lesions of the bacterial DNA (e.g., double-strand breaks) bind RecBCD enzyme. This binding enables the enzyme to begin to degrade the bacterial chromosome, but simultaneously prevents its degradative action on the ends of minor DNA species, such as unprotected infecting phage chromosomes. Degradation of the chromosomal DNA, which occurs during the early postirradiation period, ceases about 60 min after gamma ray exposure. The reappearance of the nucleolytic action of RecBCD enzyme on T4 2 DNA accompanies the cessation of degradation of bacterial DNA. Both, this cessation and the reappearance of the nucleolytic action of RecBCD enzyme on T4 2 DNA depend on a functional recA gene product. These results suggest that postirradiation DNA degradation is controlled by the recA-dependent removal of RecBCD enzyme from the damaged chromosome. By making use of the temperature-sensitive mutant recB270, we showed that RecBCD-mediated repair of gamma ray-induced lesions occurs during the early postirradiation period, i.e. during postirradiation DNA degradation. It is shown that the RecD subunit of RecBCD enzyme also participates in this repair.  相似文献   

17.
Summary We have isolated a plaque-forming derivative of phage Mu which carries a determinant for ApR. The biological properties of this MuAp phage are similar to those of normal Mu. Its genome contains a 1.1 kb substitution where Mu DNA from the right end of the G region has been replaced by a similar length of DNA from the transposon Tn3. This fragment of Tn3 DNA carries the ApR gene, but is no longer capable of independent transposition.  相似文献   

18.
Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl2, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From 32P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).  相似文献   

19.
The influence of water-soluble cationic meso-tetra-(4?N-oxyethylpyridyl)porphyrin (H2TOEPyP4) and it’s metallocomplexes with Ni, Cu, Co, and Zn on hydrodynamic and spectral behavior of DNA solutions has been studied by UV/Vis absorption and viscosity measurement. It was shown that the presence of planar porphyrins such as H2TOEPyP4, NiTOEPyP4, and СuTOEPyP4 leads to an increase in viscosity at relatively small concentrations, and then decrease to stable values. Such behavior is explained by intercalation of these porphyrins in DNA structure because the intercalation mode involves the insertion of a planar molecule between DNA base pairs which results in a decrease in the DNA helical twist and lengthening of the DNA. Further decrease of viscosity is explained by the saturation intercalation sites and occurs outside the binding mode. But, in the case of porphyrins with axial ligands such as CoTOEPyP4 and ZnTOEPyP4, the hydrodynamic parameters decrease, which is explained by self-stacking of these porphyrins in DNA surface. This data are proved by spectral measurements. The results obtained from titration experiments were used for calculation of binding parameters: the binding constant K b and the number of binding sites per base pair n. Obtained data reveal that K b varies between 3.4 and 5.4?×?106?M?1 for a planar porphyrins, a range typical for intercalation mode interactions, and 5.6?×?105?M?1 and 1.8?×?106?M?1 for axial porphyrins. In addition, the exclusion parameter n also testifies that at intercalation, (n~2) the adjacent base pairs are removed to place the planar molecules, and for outside binders to pack on the surface needs too few places (n~0.5–1). It is apparent that the binding is somewhat stronger at intercalation. The viscometric and spectrophotometric measurements are in good agreement.  相似文献   

20.
Summary A strain of Haemophilus influenzae, called hpm - inhibits the growth of phage HP1c1 but not S2. This inhibition is overcome by HP1c1ph mutants. Phage HP1c1 adsorbs normally to hpm - cells but only a small fraction of infected cells produce phage with a normal burst size or become lysogenic. When hpm - strains lysogenic for HP1c1 are induced, 100% of the cells yield phage. There is no degradation of phage DNA after infection of hpm - cells and HP1c1 can normally grow when its DNA is introduced into hpm - by transfection. The most probable explanation is that in hpm - cells the penetration of phage DNA is blocked. The hpm - property behaves as as unstable mutation.  相似文献   

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