Different effects of polylysine and polyarginine on the transition to a condensed state of DNA in polyethyleneglycol/salt solution.

Circular dichroism spectroscopy has been used to investigate the influence of polylysine and polyarginine on the transition to a condensed state of DNA brought about by high concentrations of polyethyleneglycol and salt. From the dependence on DNA concentration of the CD signals, the anomalous CD of free DNA in polyethyleneglycol/salt solution was attributed to the intermolecular association of DNA molecules. The CD spectral changes in polyethyleneglycol/salt solution of the DNA - polylysine complex were indistinguishable from those of free DNA while the DNA-polyarginine complex suffered much smaller spectral changes as compared with free DNA, at low DNA concentrations where time-independent CD spectra were observed in polyethyleneglycol/salt solution for both the complexed and free DNA. The repression of the spectral change by the latter complex was more remarkable at higher ratios of polyarginine to DNA. The facts indicate that, whereas polylysine binding has little influence on the intermolecular structural transition of double-stranded DNA into a compact molecular configuration in polyethyleneglycol/salt solution, polyarginine binding has an effect of inhibiting the transition.     

Histone and non-histone proteins from rabbit mammary gland at late pregnancy and early lactation

The content of non-histone protein in epithelial cell chromatin from rabbit mammary gland increased during late pregnancy and early lactation up to 30%, while the histone content remained unchanged. No qualitative changes, as judged by polyacrylamide-gel electrophoresis, were found during accumulation of non-histone proteins at the onset of lactation. The chromatin isolated at lactation, assayed either by polylysine or Toluidine Blue binding, contained slightly more available DNA-phosphate than the chromatin isolated at pregnancy.     

Structure and properties of rat thymus deoxyribonucleoprotein. Solubility properties and effects of precipitation

1. Deoxyribonucleoprotein was prepared from rat thymus and was studied chiefly by the method of electric birefringence. The birefringence depends on the electrical and optical properties of the molecules and on their volume; the decay time of birefringence (after the removal of the electric field) depends on molecular length. 2. A comparison was made of the properties of deoxyribonucleoprotein redissolved after precipitation in 0.15m-sodium chloride with those of the original material, the main object being to find if structural changes have occurred in the former. As a preliminary, the dependence of the solubility of the deoxyribonucleoprotein on the concentration of added salt was investigated, and the birefringence properties were also studied after the addition of sodium chloride at low concentrations, after the alteration of pH and after dialysis. 3. It was found that precipitation of deoxyribonucleoprotein occurs in two fractions, beginning at about 0.4mm-sodium chloride. The solubility is minimal at about 0.15m-sodium chloride. 4. The electric birefringence and decay time of the deoxyribonucleoprotein decrease with increasing concentration of added sodium chloride, and the birefringence also decreases after dialysis. Prolonged dialysis leads to precipitation. 5. The properties of deoxyribonucleoprotein redissolved after precipitation with 0.15m-sodium chloride differ considerably from those of the original deoxyribonucleoprotein. This is attributed to some type of disorganization process occurring during precipitation. It is concluded that the organization of the original structure is very specific.     

Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides.

In a previous report (M.S. Wadhwa et al. (1997) Bioconjugate Chem. 8, 81-88), we synthesized a panel of polylysine-containing peptides and determined that a minimal repeating lysine chain of 18 residues followed by a tryptophan and an alkylated cysteine residue (AlkCWK18) resulted in the formation of optimal size (78 nm diameter) plasmid DNA condensates that mediated efficient in vitro gene transfer. Shorter polylysine chains produced larger DNA condensates and mediated much lower gene expression while longer lysine chains were equivalent to AlkCWK18. Surprisingly, AlkCWK18 (molecular weight 2672) was a much better gene transfer agent than commercially available low molecular weight polylysine (molecular weight 1000-4000), despite its similar molecular weight. Possible explanations were that the cysteine or tryptophan residue in AlkCWK18 contributed to the DNA binding and the formation of small condensates or that the homogeneity of AlkCWK18 relative to low molecular weight polylysine facilitated optimal condensation. To test these hypotheses, the present study prepared AlkCYK18 and K20 and used these to form DNA condensates and conduct in vitro gene transfer. The results established that DNA condensates prepared with either AlkCYK18 or K20 possessed identical particle size and mediated in vitro gene transfer efficiencies that were indistinguishable from AlkCWK18 DNA condensates, eliminating the possibility of contributions from cysteine or tryptophan. However, a detailed chromatographic and electrospray mass spectrometry analysis of low molecular weight polylysine revealed it to possess a much lower than anticipated average chain length of dp 6. Thus, the short chain length of low molecular weight polylysine explains its inability to form small DNA condensates and mediate efficient gene transfer relative to AlkCWK18 DNA condensates. These experiments further emphasize the need to develop homogenous low molecular weight carrier molecules for nonviral gene delivery.     

Arrangement of histones along DNA in chromosomal deoxyribonucleoprotein lacking histone F1

In deoxyribonucleoprotein from which histone F1 and most nonhistone proteins were removed by treatment with tRNA in the presence of Mg²⁺, one can find very long stretches of completely free DNA (average length of about 4×10³ base pairs). They alternate with stretches of DNA (16×10³ base pairs) which are nearly uniformly covered with the other four histones.     

Studies on chromatin

Chromatin lacking histone H1 was found by electron microscopy to contain 'beaded' deoxyribonucleoprotein fibers. Adjacent beads are connected with each other by threads having a DNA-like appearance. At least some of threads are shown to be free DNA stretches. Average length and the content of free DNA stretches in histone H1-depleted chromatin depends on the ionic conditions of the medium. The appearance of individual beads is similar to that of chromatin subunits or v-bodies [1] in the original chromatin. Thus, in agreement with the X-ray data [2], histone H1 apparently is not required for maintenance of a compact state of DNA in chromatin subunits.     

Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein

Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at approximately 120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.Images     

Pulse radiolysis and spectral studies of the interaction of cetylpyridinium chloride and Methylene Blue with connective-tissue glycosaminoglycans and related compounds

1. Hydrated electrons produced by pulse radiolysis were used to study the interaction of polyanionic glycosaminoglycans and related compounds with the counterions Methylene Blue and cetylpyridinium chloride. 2. The effect of added salt (potassium chloride) on the interaction indicates that the relative binding affinities, with respect to the types of anionic site present, increases for both counterions in the order CO(2) (-)相似文献   

Structural organization of the bacterial nucleoid using endonucleolysis

The fragmentation of bacterial deoxyribonucleoprotein (bDNP) in the spheroplasts of Escherichia coli, Serratia marcescens, Pseudomonas fluorescens and Micrococcus luteus by bacterial intracellular Ca2+ or Ca2+, Mg2+-dependent endonucleases in situ was studied. An electrophoresis of the extracted nuclease-split bDNP revealed the presence of high molecular weight (nuclease-resistant) and low molecular weight multiple fragments (100--120 nucleotide pairs). The electrophoretic mobility of the smallest nuclease-split DNA fragments in all bacterial species under study was similar, indicating the orderly structure of bDNP. Two total fractions whose electrophoretic mobility corresponded to that of the histones H2a and H2b from calf thymus were prevalent in the spectrum of acid-soluble bDNP proteins of gram-negative species. The heterogeneity of DNP with respect to its sensitivity to nucleases, is interaction with membranes and protein distribution pattern were revealed by treatment of the bacterial nucleoid with endogenous endonucleases, which probably reflects differences in the functional state of individual sites of the genome.     

Fractionation of chromatin by differential solubility in dilute salt.

Chromatin prepared from the livers of rats was fractionated on the basis of solubility in dilute NaCl. Neither of the fractions obtained was enriched in newly synthesized DNA. The salt-soluble fraction had a higher protein content (usually up to 50%) relative to the DNA, and contained 72% or more of the rapidly synthesized RNA. This RNA was found to be complexed with the salt-soluble deoxyribonucleoprotein, not merely co-solubilized with it. Also, polylysine-binding studies showed that about 70% or more of the nucleic acid phosphates were accessible as compared to about 40% in the unfractionated chromatin. These properties suggested that the soluble fraction was enriched in activity transcribed chromatin. In contrast molecular hybridization studies showed that the complexity of the DNA and its homology with cDNA transcribed from rat-liver polysomal mRNA were the same as those of DNA from unfractionated chromatin, or from the salt-insoluble fraction. This suggests that the criteria commonly accepted as distinguishing between euchromatin and heterochromatin in vitro are not invariably valid.     

DNA synthesis in circulating erythroblasts of anemic duck. Isolation and properties of nuclear and cytoplasmic-nonmitochondrial DNA.

In the circulating blood of anemic ducks, 5% of all erythroid cells synthesize DNA. Immature erythroblasts, at all stages of differentiation, synthesize DNA although to a varying degree, while reticulocytes and erythrocytes do not. In the erythroid cell population labeled in vitro 2 h with 32Pi, half of the labeled DNA sediments as small-molecular-weight molecules, suggesting that these molecules fail to integrate into the high-molecular-weight components. Labeled DNA is found in the cytoplasmic postmitochondrial fractions and it is in a form of deoxyribonucleoproteins which cosediment with ribosomes as well as subribosomal particles in sucrose gradients. However, fixation with HCHO and centrifugation to equilibrium in CsCl gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA-dissociated ribosomes, the deoxyribonucleoprotein particles cosediment with ribosomes as well as subribosomal particles in sucorse gradients. However, fixation with HCHO and centrifugation to equilibrium in CsCl gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA-dissociated ribosomes, the deoxyribonucleoprotein particles cosdeiment with ribosomal subunits in such a way that the larger the particle, the larger the molecular weight of the DNA cosedimenting with it. The specific radioactivity of the cytoplasmic ribosome-derived and postribosomal-particle-derived DNAs and the small molecular-weight nuclear DNA is similar and 10-20-fold higher than that of the bulk nuclear DNA. The former three DNA species sediment between 4-14 S. It is concluded that the cytoplasmic nonmitochondrial DNA species are of the nuclear origin. Less than 0.5% of the total cellular nonmitochondrial DNA can be purified from the nucleus and the cytoplasm as fast-labeled small-molecular-weight components. All of the cellular nonmitochondrial DNA species band at the same mean buoyand density in Cs2SO4/urea gradients. All behave as native structures in hydroxyapatite and contain less than 5% of their length as single-stranded regions.     

Cytochemical properties of euchromatin and heterochromatin

Two classic cytochemical tests, the Feulgen-Schiff reaction and Toluidine Blue basophilia, have been employed for investigating the differential characteristics of heterochromatin and euchromatin. Differences have been detected in the Feulgen hydrolysis kinetics, the Feulgen absorption spectrum, the image analysis of Feulgen-stained material, and the binding of Toluidine Blue under ordinary and Mg2+ competitive staining conditions. The differences are assumed to be a function of the composition and stereo-arrangement of the DNA and DNA-protein complexes present in these chromatin types and are possibly associated with physiological activities whose whole meaning is far from being clear. Differences in optical retardations in Toluidine Blue-stained material were also found. These are interpreted as being due to chromatin packing state and selective removal of histones promoted by the acetic acid-ethanol fixative.     

Thermal denaturation of calf thymus DNA: existence of a GC-richer fraction

In 2.5 x 10(-4)M EDTA buffer, the derivative melting curve of calf thymus DNA shows a major band at 47 degrees with a shoulder at about 54 degrees . The fraction of melting area of this shoulder is about 13%. For reconstituted polylysine-calf thymus DNA complexes, in addition to the melting of free DNA regions at about 50 degrees (T(m)) there is another melting at about 106 degrees (T(m)) of polylysine-bound regions. The melting band of the complex at T(m) is not symmetrical. As more polylysine is bound to DNA the melting amplitude is diminished greatly on the major band at 47 degrees but only slightly on the shoulder at 54 degrees . The insensitivity of this shoulder appears to result from the existence of a 13% fraction of calf thymus DNA containing 55% GC. It is not favorably bound by polylysine. It remains in the supernatant after centrifugation and melts at about 54-56 degrees . This conclusion is further supported by two facts: the reconstitution method provides a condition for selective binding of polylysine to AT-rich DNA, and it yields a fully symmetric melting band at T(m) for complexes of polylysine with homogeneous bacterial DNA such as the one from M. luteus.     

Helix-coil transition in nucleoprotein: renaturation of polylysine-DNA and polylysine-nucleohistone complexes

Thermal denaturation and renaturation of directly mixed and reconstituted polylysine–DNA, directly mixed polylysine–nucleohistone complexes, and NaCl-treated nucleohistones in 2.5 × 10^?4 M EDTA, pH 8.0 have been studied. At the same input ratio of polylysine to DNA, the percent of renaturation of free base pairs in a directly mixed polylysine–DNA complex is higher than that in a reconstituted complex. For a directly mixed complex, the renaturation of free base pairs is proportional to the fraction of DNA bound by polylysine or inversely proportional to the sizes of free DNA loops. A of large amount of renaturation of free base pairs has also been observed for 0.6 M and 1.6 M NaCl-treated nucleohistones. The binding of polylysine to nucleohistone enhances the renaturation of histone-bound base pairs. The percent of renaturation of polylysine–bound base pairs is high and is approximately independent of the extent of binding on DNA by polylysine. This is true in polylysine–DNA complexes prepared either by reconstitution or by directly mixing. It also applies for polylysine–nucleohistone complexes. The model where polylysine-bound base pairs collapse at T_m′ with two complementary strands still bound by polylysine is favored over the model where polylysine is dissociated from DNA during melting. The low renaturation of histone-bound base pairs in nucleo-histone indicates that either histones do not hold two complementary strands of DNA tightly or that histones are fully or partially dissociated from DNA when the nucleo-histone is fully denatured.     

Repair of lesions induced by bruneomycin in DNA of isolated mitochondria from the mature eggs of the teleost fish Misgurnus fossilis

Addition of a radiomimetic antibiotic bruneomycin (Streptonigrin) to isolated mitochondria from mature quiescent oocytes of the teleost fish loach Misgurnus fossilis leads to the induction of unscheduled synthesis of mitochondrial DNA. Most of the newly synthesized DNA has the sedimentation properties of open circles and up to 15% of the label is present in the fraction of the covalently closed-circular molecules. The size of the newly synthesized DNA stretches determined from the bouyant shift of DNA labeled with 5-bromouracil and [3H]dAMP and sonicated to fragments of different molecular weight, was found to be equal to about 1000 nucleotides for the labeled covalently closed circles and to about 2000 nucleotides for the labeled open-circular DNA. Experiments with the centrifugation of non-sheared and sonicated 5-bromouracil and [3H]dAMP-labeled mitochondrial DNA (mtDNA) in alkaline CsCl density gradients provided evidence of a covalent linkage between newly-synthesized stretches and the parental DNA strands. It is concluded from these data that the unscheduled mtDNA synthesis induced by bruneomycin does at least in part represent mtDNA repair synthesis.     

Free radical-induced chitosan depolymerized products protect calf thymus DNA from oxidative damage

Low molecular weight chitosan (LMWC) and chitooligosaccharides (COs), obtained by persulfate-induced depolymerization of chitosan showed scavenging of OH. and O2.- radicals and offered protection against calf thymus DNA damage. Over 85% inhibition of free radicals and DNA protection were observed. LMWC (0.05 micromol) showed a strong inhibitory activity compared to COs (3.6 micromol). Further, LMWC showed calf thymus DNA condensation reversibly giving stability, as evident from CD, TEM and melting curves (Tm). A fluorescence study suggests the binding of LMWC in the minor groove, forming H-bonds to the backbone phosphates without distorting the double helix structure.     

Detection of hybrid formation between peptide nucleic acids and DNA by fluorescence polarization in the presence of polylysine.

A new method for the detection of PNA/DNA hybrids is presented. In this method, short PNA probes (9-13 mer) are labeled with a fluorescent dye and allowed to hybridize to target DNA molecules. A cationic polyamino acid, such as polylysine, is then added to the reaction mixture, whereupon the DNA molecules bind electrostatically to this polycation. The PNA probes, which are uncharged or may carry only a small charge due to the fluorescent dye, do not bind to polylysine unless hybridized to the negatively charged DNA target. The binding of the labeled PNA/DNA hybrid to the high-molecular-weight polymer leads to a significant change in the rotational correlation time of the fluorophore attached to the PNA. This can be conveniently detected by measuring the fluorescence polarization of the latter. The method is completely homogeneous because no separation of free from bound PNA probe is required. The hybridization and dehybridization reactions can be followed in real time. The method has been applied to the typing of single-nucleotide polymorphisms in PCR products.     

Nature of RNA synthesis on embikhin-damaged templates

Embichin, a bifunctional alkylating mutagenic agent, inhibits RNA-synthesizing capacity of DNA and chromatin. The template capacity for decreasing the embichin-injured chromatin is caused by substantial reduction in the number of RNA molecules synthesized by this template. When DNA extracted from embichin-injured chromatin is used as a template, its template restriction is accounted for by an approximately 5-fold decrease in the RNA average molecular weight. The deoxyribonucleoprotein complexes reconstituted from embichin-injured DNA and control chromatic proteins had a lower template capacity compared with that of DNP reconstituted from control DNA. It is concluded that both links between DNA and proteins and DNA injuries are responsible for the inhibition of genome template capacity.     

High diversity in DNA of soil bacteria.

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.