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1.
With the use of highly competent Haemophilus influenzae cells, it was possible to demonstrate the uptake of heterologous DNAs. However, these DNAs, as expected, were only 1% or less as effective when competing for uptake with Haemophilus DNA. Escherichia coli DNA was removed from solution by competent cells to the extent expected if all the E. coli DNA particles contained at least one uptake recognition signal. The data were consistent with a model in which there was one uptake signal per 20 X 10(6) to 30 X 10(6) daltons of E. coli DNA. Since H. influenzae DNA has many more recognition signals, approximately one per 2 X 10(6) daltons (Danner et al., Gene 77:311-318, 1980; K. Vogt and S. H. Goodgal, submitted for publication), it has been suggested that the slower rate of E. coli DNA binding and the so-called specificity of Haemophilus DNA binding are due to the number of recognition signals per molecule of DNA as well as the nature of the DNA receptor (Vogt and Goodgal, submitted for publication). The specificity of native H. influenzae DNA binding does not apply to the uptake of denatured DNA in the transforming system (low pH) for denatured DNA. 相似文献
2.
The binding of pancreatic ribonuclease-A by denatured DNA, native DNA, poly-dA, and poly-dT, has been studied by a gel filtration method. With denatured DNA at pH 7.5, ionic strength 0.053M, there is one binding site per 12 nucleotides and the equilibrium binding constant per site is 9.7 × 104 l./mole. The binding constant increases by a factor of 8 as the pH is decreased from 8 to 7. The strength of the binding of denatured DNA increases with decreasing ionic strength. At pH 7.5, native DNA binds about ? as strongly as does denatured DNA. The binding affinity increases in the order poly-dA, denatured DNA, and poly-dT. These results support the view that the binding of denatured DNA involves both electrostatic interactions between the negatively charged polynucleotide and the positively charged protein, and an interaction of the protein with a pyrimidine residue of the denatured DNA, and thus that the binding is basically similar to that between RNAse and its substrate RNA. 相似文献
3.
Ghosh J Basu A Pal S Chowdhuri S Bhattacharya A Pal D Chattoraj DK DasGupta C 《Molecular microbiology》2003,48(6):1679-1692
Bacterial ribosomes or their 50S subunit can refold many unfolded proteins. The folding activity resides in domain V of 23S RNA of the 50S subunit. Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo. The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro. The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact. The DnaK binding influenced the protein folding activity of domain V modestly. Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit. DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity. 相似文献
4.
Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA 总被引:14,自引:0,他引:14
We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites. 相似文献
5.
Photodynamic Action on Native and Denatured Transforming Deoxyribonucleic Acid from Haemophilus influenzae
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The photodynamic inactivation of native or denatured transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae is described. The inactivation at the same pH was higher for denatured than native DNA. At acidic pH, the inactivation both for native and denatured DNA was faster than at alkaline pH. The guanine content of photoinactivated native DNA at neutral pH was less than untreated DNA. The inactivation of biological activity was more extensive than the alteration of guanine. The absorption spectrum of photoinactivated native or denatured DNA was only slightly different than the control DNA at the different experimental conditions. 相似文献
6.
Neurofilaments were isolated from porcine spinal cord and separated into their subunit proteins (68 Kd NFP, 145 Kd NFP, 200 Kd NFP) by ion exchange chromatography on DEAE-cellulose in 6 M urea. The individual proteins were reacted with total rRNA from Ehrlich ascites tumor cells and the reaction products analysed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. All three proteins interacted with rRNA with a preference for 18S rRNA. Competition experiments with native and heat-denatured calf thymus DNA showed that the affinities of the 68 Kd and 145 Kd NFPs were considerably higher for denatured DNA than for rRNA and that native DNA was only a weak competitor. The binding of the 200 Kd NFP to rRNA was unaffected by native and by denatured DNA. When denatured DNA was reacted with a mixture of the 68 Kd and 145 Kd NFPs, the two proteins interacted independently with the nucleic acid, giving rise to two different populations of deoxyribonucleoprotein particles. This segregation is the result of the cooperative interaction of the neurofilament proteins with single-stranded DNA. It could not be observed with rRNA or bacteriophage MS2 RNA. The results clearly show that the 68 Kd and 145 Kd NFPs are single-stranded RNA- and DNA-binding proteins, whereas the 200 Kd NFP seems to be only a single-stranded RNA-binding protein. 相似文献
7.
A lead-absorbing protein with superoxide dismutase activity from Streptomyces subrutilus 总被引:3,自引:0,他引:3
A lead binding protein was purified from the culture filtrate of Streptomyces subrutilus P5. The subunit and native molecular weights were estimated to be 28 and 55 kDa, respectively, indicating that the protein was composed of two identical subunits. The inhibition pattern, the metal content analysis and the EPR spectrum confirmed that the protein was a superoxide dismutase containing Fe and Zn (FeZnSOD). The protein precipitated immediately upon mixing with lead ions and the saturation number of lead ions was about 1100 lead atoms per subunit. Using this property, lead ions could be effectively removed from solutions. 相似文献
8.
Proflavine formed a complex with transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae, with optimal formation at a ratio of proflavine to DNA of 0.06. The rate of dissociation of the complex by dialysis increased in the order: native, denatured, renatured DNA. The transforming activity of the DNA was reduced by its interaction with proflavine. This inactivation was dependent on the physical state of the DNA, the proflavine concentration, and the temperature. DNA that had been denatured and renatured was most sensitive; native DNA was much less sensitive. The inactivation remained after dialysis and was stable to prolonged storage. It is concluded that the inactivation of transforming DNA by proflavine takes place by a mechanism different from that of DNA-proflavine complex formation. 相似文献
9.
Native and denatured calf thymus DNA, and homopolyribonucleotides were compared with respect to chromium and protein binding after an in vitro incubation with rat liver microsomes, NADPH, and chromium(VI) or chromium(III). A significant amount of chromium bound to DNA when chromium(VI) was incubated with the native or the denatured form of DNA in the presence of microsomes and NADPH. For both native and denatured DNA the amount of protein bound to DNA increased with the amount of chromium bound to DNA. Denatured DNA had much higher amounts of chromium and protein bound than native DNA. There was no interaction between chromium(VI) and either form of DNA in the absence of the complete microsomal reducing system. The binding of chrornium(III) to native or denatured DNA was small and relatively unaffected by the presence of microsomes and NADPH. The binding of chromium and protein to polyriboadenylic acid (poly(A)), polyribocytidylic acid (poly(C), polyri-boguanylic acid (poly(G)) and polyribouridylic acid (poly(U)) was determined after incubation with chromium(VI) in the presence of microsomes and NADPH. The magnitude of chromium and protein binding to the ribo-polymers was found to be poly(G) ? poly(A) ? poly(C) ? poly(U). These results suggest that the metabolism of chromium(VI) is necessary in order for chromium to interact significantly with nucleic acids. The metabolically-produced chromium preferentially binds to the base guanine and results in DNA-protein cross-links. These findings are discussed with respect to the proposed scheme for the carcinogenicity of chromium(VI). Keywords: DNA-protein cross-links — Chromium-guanine interaction-Microsomal reduction of chromate 相似文献
10.
B E Magun 《Experimental cell research》1976,103(1):219-231
The cytoplasmic DNA-binding proteins of Physarum polycephalum were recovered by chromatography of cytosol extracts on sequential columns of native and denatured calf thymus DNA-cellulose. 5.4% of the total cytosol protein was bound to native DNA-cellulose, while 4.4% was bound to denatured DNA-cellulose. Stepwise salt gradient elution of the columns separated the DNA-binding proteins into 9 fractions which were analysed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Several hundred discrete polypeptide bands were identified, with many more high molecular weight polypeptides (greater than 100 000 D) binding to native than to denatured DNA. Continuous in vivo labelling of microplasmodia in KH2[32P]O4 and [3H]leucine was used to determine which of the DNA-binding proteins were phosphorylated, and to approximate their phosphorus content. About 30–40 phosphoproteins were resolved among the DNA-binding proteins. Most phosphoproteins contained less than 3 phosphates per polypeptide, but a small number of low molecular weight phosphoproteins (less than 50 000 D) contained from 5 to 10 phosphates per polypeptide. The majority of high molecular weight DNA-binding phosphoproteins bound to native DNA and were eluted with 0.25 M NaCl. As a group, the DNA-binding proteins were enriched in protein-bound phosphorus when compared with the cytosol proteins which did not bind to DNA. The phosphorus content of the cytoplasmic DNA-binding proteins was similar to that of the acidic nuclear proteins. 相似文献
11.
Grigorios A. Papadakos Cynthia M. Dupureur 《Journal of biological inorganic chemistry》2011,16(8):1269-1278
Understanding the roles of metal ions in restriction enzymes has been complicated by both the presence of two metal ions in
many active sites and their homodimeric structure. Using a single-chain form of the wild-type restriction enzyme PvuII (scWT) in which subunits are fused with a short polypeptide linker (Simoncsits et al. in J. Mol. Biol. 309:89–97, 2001), we have characterized metal ion and DNA binding behavior in one subunit and examined the effects of the linker on dimer
behavior. scWT exhibits heteronuclear single quantum coherence NMR spectra similar to those of native wild-type PvuII (WT). For scWT, isothermal titration calorimetry data fit to two Ca(II) sites per subunit with low-millimolar K
ds. The variant scWT|E68A, in which metal ion binding in one subunit is abolished by mutation, also binds two Ca(II) ions in
the WT subunit with low-millimolar K
ds. When there are no added metal ions, DNA binding affinity for scWT is tenfold stronger than that of the native WT, but tenfold
weaker at saturating Ca(II) concentration. In the presence of Ca(II), scWT|E68A binds target DNA similarly to scWT, indicating
that high-affinity substrate binding can be carried energetically by one metal-ion-binding subunit. Global analysis of DNA
binding data for scWT|E68A suggests that the metal-ion-dependent behaviors observed for WT are reflective of independent subunit
behavior. This characterization provides an understanding of subunit contributions in a homodimeric context. 相似文献
12.
13.
The binding properties of protein uH2A and histone H2A to DNA were investigated by filter binding assays. Both proteins revealed similar affinity for native and denatured DNA. Competition with increasing amounts of repetitive and nonrepetitive DNA has shown that protein uH2A binds selectively to nonrepetitive sequences. When poly d(A-T) was used as a competitor, uH2A bound to this polynucleotide with much greater affinity than histone H2A. These findings suggest a selective binding to regulatory A-T rich intergenic sequences in native DNA. 相似文献
14.
Interactions of competent Streptococcus sanguis (Wicky) cells with native or denatured, homologous or heterologous deoxyribonucleic acids.
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J M Ranhand 《Journal of bacteriology》1980,142(2):568-580
Competent cell-deoxyribonucleic acid (DNA) interactions were examined using tritium-labeled homologous or heterologous native or denatured DNAs and competent Streptococcus sanguis Wicky cells (strain WE4). The DNAs used were extracted from WE4 cells, Escherichia coli B cells, and E. coli bacteriophages T2, T4, T6, and T7. The reactions examined were: (i) total DNA binding, (ii) deoxyribonuclease-resistant DNA binding, and (iii) the production of acid-soluble products from the DNA. Optimal temperatures for the reactions were as follows: reaction (i), between 30 and 40 degrees C; reaction (ii), 30 degrees C; and reaction (iii), greater than 40 degrees C. The rates for the reactions (expressed as molecules of DNA that reacted per minute per colony-forming unit) did not vary greatly from one DNA source to another. With a constant competent cell concentration and differing DNA concentrations below a saturation level (from a given source), a different but constant fraction of the added DNA was cell bound, deoxyribonuclease resistant, and degraded to acid-soluble products. In experiments where the number of competent cells was varied and the DNA concentration was held constant, again essentially the same result was obtained. The extent of reactions (i), (ii), and (iii) depended upon the numbers as well as the source of DNA molecules applied to competent cells. Calcium ion essential for native DNA-cell reactions was also found essential for denatured DNA-cell reactions. Data obtained from competition experiments lead to the conclusion that competent WE4 cells contain specific sites for native as well as denatured DNAs. 相似文献
15.
Identification of lysine residues at the binding site of bacteriophage-Pf1 DNA-binding protein. 总被引:1,自引:0,他引:1
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The accessibility of NH2 groups in the DNA-binding protein of Pf1 bacteriophage has been investigated by differential chemical modification with the reagent ethyl acetimidate. The DNA-binding surface was mapped by identification of NH2 groups protected from modification when the protein is bound to bacteriophage-Pf1 DNA in the native nucleoprotein complex and when bound to the synthetic oligonucleotide d(GCGTTGCG). The ability of the modified protein to bind to DNA was monitored by fluorescence spectroscopy. Modification of the NH2 groups in the native nucleoprotein complex showed that seven out of the eight lysine residues present, and the N-terminus, were accessible to the reagent, and were not protected by DNA or by adjacent protein subunits. Modification of these residues did not inhibit the ability of the protein to bind DNA. Lysine-25 was identified by peptide mapping as being the major protected residue. Modification of this residue does abolish DNA-binding activity. Chemical modification of the accessible NH2 groups in the complex formed with the octanucleotide effectively abolishes binding to DNA. Peptide mapping established that, in this case, lysine-17 was the major protected residue. The differences observed in protection from acetimidation, and in the ability of the modified protein to bind DNA, indicate that the oligonucleotide mode of binding is not identical with that found in the native nucleoprotein complex with bacteriophage-Pf1 DNA. 相似文献
16.
Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein. 总被引:7,自引:4,他引:3
In DNA binding-deficient mutants of Bacillus subtilis a competence-specific protein with a subunit molecular weight of 18,000 was absent. The native protein containing this subunit was purified from B. subtilis membranes by chromatography on hydroxyapatite, DEAE-cellulose, and Sephacryl S-200. This protein appeared to be complexed with a second protein of slightly lower molecular weight (17,000) and a different isoelectric point. The native protein complex (apparent molecular weight, 75,000) contained approximately equal amounts of the two polypeptides and showed a strong DNA-binding activity. Incubation of the complex with plasmid and bacteriophage DNA revealed nuclease activity, specifically directed toward double-stranded DNA. Predominantly single-stranded nicks and a limited number of double-stranded breaks were introduced in the presence of Mg2+ ions. In the presence of Mn2+ ions the complex produced low-molecular-weight breakdown products from the DNA. 相似文献
17.
Relationship Between Competence for Transformation of Bacillus subtilis with Native and Single-Stranded Deoxyribonucleic Acid 总被引:1,自引:1,他引:0
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The response of populations of Bacillus subtilis to both native deoxyribonucleic acid (DNA) and denatured DNA was investigated at maximal competence and at various times during the development of compentency. The results indicate that competence for transformation with native and denatured DNA increases and decreases simultaneously. Competition occurs between native and single-stranded DNA during transformation, and the same cells in a population can be doubly transformed by DNA molecules of both configurations. 相似文献
18.
The Mac-1 and p150,95 beta 2 integrins bind denatured proteins to mediate leukocyte cell-substrate adhesion. 总被引:10,自引:0,他引:10
G E Davis 《Experimental cell research》1992,200(2):242-252
Activated monocytic cells and neutrophils adhere to substrates coated with a wide variety of proteins including albumins, catalase, casein, and various extracellular matrix proteins. This adhesion can be specifically inhibited by antibodies directed to the beta 2 integrin subunit. This adhesion to protein substrates shares some similarities with two known protein-protein recognition systems with little apparent binding specificity, namely, the interactions of heat shock proteins and histocompatibility antigens with denatured proteins or peptides. Cell adhesion and affinity chromatography experiments were performed to test the hypothesis that monocytes and neutrophils adhere to and migrate on protein substrates due to the presence of cell surface receptors that recognize common protein structures such as denatured protein epitopes. Adhesion experiments revealed that activated monocytic cells adhere more rapidly and extensively on substrates coated with denatured protein versus native protein. Both adhesion and migration on such substrates in vitro was dependent on beta 2 integrins since blocking antibodies completely interfered with these cellular responses. Affinity chromatography experiments revealed that the Mac-1 and p150,95 integrins could be isolated from monocyte-differentiated HL-60 cells or neutrophils on a denatured protein-Sepharose column. Much greater yields of the receptors were obtained on a denatured versus native protein Sepharose column. The binding of these receptors was specific in that the LFA-1 beta 2 integrin did not bind to the denatured protein column. These data provide evidence that the adhesion of activated monocytes and neutrophils to many protein substrates in vitro is due to the ability of Mac-1 and p150,95 to directly bind to denatured proteins. A model of leukocyte adhesion and invasion whereby activated leukocytes denature extracellular proteins during diapedesis, making them suitable for recognition by beta 2 integrins, is proposed. 相似文献
19.
Renaturation of DNA in the presence of ethidium bromide 总被引:1,自引:0,他引:1
The rate of renaturation of T2 DNA has been studied as a fuction of ethidium bound per nucleotide of denatured DNA. The Binding constants and number of binding sites for ethidium have been determined by spectral titration for denatured DNA at 55, 65, and 75°C and for native DNA at 65°C in 0.4M Na+. The rate of renaturation of T2 DNA was found to be independentof ethidium binding up to 0.03 moles per mole of nucleotide. Above 0.03 moles, the rate drops off precipitously approaching zero at 0.08 and 0.06 moles bound ethidium per nucleotide at 65°C respectively. A study was also made of the use of bound ethidium fluorescence as a probe for monitoring DNA renaturation reactions. 相似文献
20.
Polarographic investigation of binding of Cu++ and Cd++ by DNA 总被引:1,自引:0,他引:1
The equilibrium binding constants of Cd++ and Cu++ to native and denatured calf thymus DNA were determined polarographically. The binding constants are an exponential function of the potential at the binding site and as such they vary with ionic strength and with the charge on the DNA molecule. The correlation between the fraction of sites occupied by heavy metal ions and between the thermal stability of DNA in solution is discussed. 相似文献