Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria

1. Nucleic acids were released from Escherichia coli by lysing with tri-iso-propylnaphthalene sulphonate and 4-aminosalicylate and then extracting with a phenol-cresol mixture. 2. Nucleic acids were similarly released from Bacillus subtilis after initial treatment with lysozyme. 3. DNA was sedimented after careful precipitation with m-cresol or 2-butoxyethanol (0.1-0.12vol.) in the presence of 20% sodium benzoate. 4. Contaminating ribosomal RNA was removed by precipitation in the presence of 4m-sodium chloride or by extracting DNA with an acetate-butyrate mixture, in which RNA is insoluble. 5. The DNA from B. subtilis has a transforming ability of 0.3-0.6% for the tryptophan marker. 6. Ribosomal RNA was then precipitated with rapidly labelled RNA by the addition of an equal volume of 2-butoxyethanol. 7. There was good separation of the nucleic acids from protein and polysaccharides.     

Interactions between the lysine-rich histone F1 and deoxyribonucleic acid

1. The interactions of the lysine-rich histone F1 with DNA have been studied at various histone to DNA ratios, in water and in the presence of uni- and bi-valent cations. In water only, histone F1, even in fourfold excess, is unable to precipitate all the DNA. In 0.14m-sodium chloride, 0.8mg. of histone F1 is required to precipitate 1mg. of DNA, whereas in 0.07m-magnesium chloride only 0.4mg. is required. 2. Bivalent cations are also shown to be more effective in dissociating the DNA-histone complex. Histone F1 can be selectively removed from deoxyribonucleoprotein with 0.1m-magnesium chloride. 3. The precipitation of DNA by histone F1 is a reversible process and the complex can be taken in and out of solution by changing the ionic environment. 4. The bearing of these results on the observed ability of various DNA-histone complexes to act as templates for RNA synthesis is discussed.     

The dissociation of chicken erythrocyte deoxyribonucleoprotein and some properties of its partial nucleoproteins

Histones were completely dissociated from their native complex with DNA in 2.0m-sodium chloride. Histone fractions IIb, V and I were dissociated in 1.2m-sodium chloride, fractions V and I in 0.7m-sodium chloride and fraction I in 0.45m-sodium chloride. Repeated extraction of partial dRNP (deoxyribonucleoprotein) preparations with sodium chloride of the same concentration as that from which they were prepared resulted in release of histones that previously had remained associated with the DNA of the complex. Gradual removal of histones from dRNP was paralleled by an improvement in solubility, a decrease in wavelength of the u.v.-absorption minimum, and a fall in sedimentation coefficient of the remaining partial dRNP. X-ray diffraction patterns of partial dRNP preparations showed that removal of histone fractions I and V from dRNP did not destroy the super-coil structure of the dRNP, but further removal of histones did. Infrared spectra of partial dRNP preparations showed that in native dRNP histone fraction I was present in the form of extended, isolated polypeptide chains, and that the other histone fractions probably contain a helical component that lies roughly parallel to the polynucleotide chains in the double helix and an extended polypeptide component that is more nearly parallel to the DNA helix axis. An analysis of the sedimentation of partial dRNP preparations on sucrose gradients showed that native dRNP consists of DNA molecules each complexed with histone fractions of all types.     

A spectrophotometric study of the denaturation of deoxyribonucleic acid in the presence of urea or formaldehyde and its relevance to the secondary structure of single-stranded polynucleotides

1. The thermal denaturation of DNA from rat liver was studied spectrophotometrically. In sodium phosphate buffers denaturation led to a single-stranded form having, at 25 degrees , about 25% of the hypochromism of the intact double helix. 2. The hypochromism of the denatured form was the same in 1mm- as in 10mm-sodium phosphate buffer and was scarcely affected by reaction with formaldehyde. The hypochromism was decreased by about 40% in the presence of 8m-urea. 3. The hypochromism of denatured DNA at low ionic strengths was about the same as that of fragments of reticulocyte ribosomal RNA that were too short to form double-helical secondary structure and about the same as that of RNA after reaction with formaldehyde. 4. The spectrum of DNA was slightly affected by the presence of 8m-urea or 4m-guanidinium chloride. The differences in the spectrum of the native and denatured forms of DNA in 0.1m-sodium phosphate buffer, in 8m-urea-10mm-sodium phosphate buffer and in 4m-guanidinium chloride-10mm-sodium phosphate buffer, pH7.6, were similar but not identical. 5. Denatured rat liver DNA appears to have no double-helical character at 25 degrees in 10mm-sodium phosphate buffer, pH7.6; increasing the buffer concentration to 0.1m leads to a more compact form in which about 40% of the residues form base pairs.     

Interactions of cytochrome c and [14C]-carboxymethylated cytochrome c with monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin

1. The interactions between cytochrome c (native and [(14)C]carboxymethylated) and monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin at the air/water interface was investigated by measurements of surface radioactivity, pressure and potential. 2. On a subphase of 10mm-or m-sodium chloride, penetration of cytochrome c into egg phosphatidylcholine monolayers, as measured by an increase of surface pressure, and the number of molecules penetrating, as judged by surface radioactivity, were inversely proportional to the initial pressure of the monolayer and became zero at 20dynes/cm. The constant of proportionality was increased when the cytochrome c was carboxymethylated or decreased when the phospholipid was hydrogenated, but the cut-off point remained at 20dynes/cm. 3. Penetrated cytochrome c could be removed almost entirely by compression of the phosphatidylcholine monolayer above 20dynes/cm. 4. With phosphatidic acid and cardiolipin monolayers on 10mm-sodium chloride the binding of cytochrome c was much stronger and cytochrome c penetrated into films nearing the collapse pressure (>40dynes/cm.). The penetration was partly electrostatically facilitated, since it was decreased by carrying out the reaction on a subphase of m-sodium chloride, and the relationship between the surface pressure increment and the initial film pressure moved nearer to that observed with phosphatidylcholine. 5. Surface radioactivity determinations showed that [(14)C]carboxymethylated cytochrome c was still adsorbed on phosphatidic acid and cardiolipin monolayers after the cessation of penetration. This adsorption was primarily electrostatic in nature because it could be prevented and substantially reversed by adding m-sodium chloride to the subphase and there was no similar adsorption on phosphatidylcholine films. 6. The penetration into and adsorption on the three phospholipid monolayers was examined as a function of the pH of the subphase and compared with the state of ionization of both the phospholipid and the protein, and the area occupied by the latter at an air/water interface. 7. It is concluded that the binding of cytochrome c to phospholipids can only be partially understood by a consideration of the ionic interaction between the components and that subtle conformational changes in the protein must affect the magnitude and stability of the complex. 8. If cytochrome c is associated with a phospholipid in mitochondria then cardiolipin would fulfil the characteristics of the binding most adequately.     

Immunoglobulin M biosynthesis. Production of intermediates and excess of light chain in mouse myeloma MOPC 104E

1. Conditions have been established for the estimation of molecular weights of proteins by analytical gel filtration and sucrose-density-gradient centrifugation in 2.5m-potassium chloride-1m-sodium chloride; Halobacterium cutirubrum polynucleotide phosphorylase, DNA-dependent RNA polymerase and RNA-dependent RNA polymerase have been studied by these methods. 2. The RNA-dependent polymerase has also been studied by density-gradient centrifugation in the absence of salt. 3. All three proteins are of unusually low molecular weight compared with similar enzymes from non-halophilic bacteria.     

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.     

Renaturation of bacteriophage phiX174 DNA-RNA hybrid: RNA length effect and nucleation rate constant

A one to one RNA-DNA hybrid was synthesized using bacteriophage φX174 DNA as a template for the Escherichia coli RNA polymerase system. The renaturation rate for the hybrid was found to vary as the square root of the molecular weight of the RNA. The RNA molecular weights were determined using a standardized dimethylsulfoxide, cesium sulfate equilibrium density gradient. The optimum nucleation rate constant for the renaturation of the hybrid in 0.4 m-sodium chloride was found to be 1.3 × 10⁵, as compared with to 1.7 × 10⁵ for DNA renaturation.     

The significance of cytochrome c redistribution during the subcellular fractionation of rat liver

1. The redistribution of mitochondrial cytochrome c during homogenization and subcellular fractionation of the liver was studied. Chromatographically homogeneous (14)C-labelled cytochrome c was added in different amounts to liver suspensions immediately before homogenization and the adsorption of radioactivity was determined in cytochrome c fractions extracted at pH4.0, first with water and then with 0.15m-sodium chloride. 2. The soluble cytochrome c remaining in the cell sap after subcellular fractionation was 7% of the calculated amount of cytochrome c passing through a soluble form during the whole process. The total amount of cytochrome c released in a soluble form and subsequently redistributed was 25-30% of the total liver cytochrome c. 3. In the standard microsomal fraction the cytochrome c extracted with water originated entirely from redistribution whereas that extracted with 0.15m-sodium chloride was 80% endogenous. In the mitochondrial fraction both cytochrome c pools were truly endogenous, so that practically none of the mitochondrial cytochrome c released to the soluble cell sap was readsorbed by the mitochondria. 4. These results support our former hypothesis that the cytochrome c extracted with 0.15m-sodium chloride at pH4.0 from the standard microsomes represents the cytochrome c newly synthesized in situ, since it does not originate from redistribution. However, the microsomal pool extracted with water cannot be an intermediate in the postulated transfer of cytochrome c from the microsomal particles to the mitochondria, since this pool arises from redistribution of mitochondrial cytochrome c.     

Isolation and partial characterization of apiogalacturonans from the cell wall of Lemna minor

1. A mild, reproducible extraction procedure, using 0.5% ammonium oxalate, was developed for the isolation of polysaccharides containing d-apiose from the cell wall of Lemna minor. On a dry-weight basis the polysaccharide fractions extracted with ammonium oxalate made up 14% of the material designated cell walls and contained 20% of the d-apiose originally present in the cell walls. The cell walls, as isolated, contained 83% of the d-apiose present in L. minor. 2. After extraction with ammonium oxalate, purified polysaccharides were obtained by DEAE-Sephadex column chromatography and by fractional precipitation with sodium chloride. With these procedures the material extracted at 22 degrees C could be separated into at least five polysaccharides. On a dry-weight basis two of these polysaccharides made up more than 50% of the material extracted at 22 degrees C. There was a direct relationship between the d-apiose content of the polysaccharides and their solubility in sodium chloride solutions; those of highest d-apiose content were most soluble. 3. All the polysaccharides isolated appeared to be of one general type, namely galacturonans to which were attached side chains containing d-apiose. The d-apiose content of the apiogalacturonans varied from 7.9 to 38.1%. The content of esterified d-galacturonic acid residues in all apiogalacturonans was low, being in the range 1.0-3.5%. Hydrolysis of a representative apiogalacturonan with dilute acid resulted in the complete removal of the d-apiose with little or no degradation of the galacturonan portion. 4. Treatment of polysaccharide fractions with pectinase established that those of high d-apiose content and soluble in m-sodium chloride were not degraded, whereas those of low d-apiose content and insoluble in m-sodium chloride were extensively degraded. When the d-apiose was removed from a typical pectinase-resistant polysaccharide, the remainder of the polysaccharide was readily degraded by this enzyme. 5. Periodate oxidation of representative polysaccharide fractions and apiogalacturonans and determination of the formaldehyde released showed that about 50% of the d-apiose molecules were substituted at either the 3- or the 3'-position.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

The effect of high ionic strength on the sedimentation properties of the biologically active component of bacterial ribonucleic acid

1. The sedimentation properties of the fraction of bacterial RNA which stimulates the incorporation of amino acids into acid-insoluble material in vitro depend on the ionic strength of the sedimentation medium. 2. The different distributions of stimulatory activity found in centrifuged linear sucrose gradients loaded with bacterial RNA and containing 0.1m- or 0.6m-sodium chloride resemble closely the different sedimentation profiles of rapidly labelled RNA observed under the same conditions. 3. These findings agree with those of previous work of a similar nature (Willson & Gros, 1964) and demonstrate that the component of bacterial RNA preparations which stimulates the incorporation of amino acids into acid-insoluble peptides in vitro is contained in their rapidly labelled fraction.     

Magnesium ions and the structure of Escherichia coli ribosomal ribonucleic acid

1. The effect of removing Mg(2+) from a purified high-molecular-weight (1.07x10(6)) fraction of Escherichia coli ribosomal RNA was examined by ultracentrifugation, thermal denaturation and optical rotation. 2. At moderate I (0.1m-sodium chloride), EDTA at 2-50mm has little effect on RNA; at low I, 0.01-0.04 (with tris as counter-ion), two boundaries appear. 3. The leading boundary, S(20,w) about 20s, is identified with the original material with counter-ion Mg(2+) (;ionic atmosphere') removed, leading to an expanded form. 4. The slow boundary, 15-16s, is associated with a further loss of Mg(2+) and a further expansion, sensitive to EDTA concentration: it is proposed that this Mg(2+) is localized on the polynucleotide chain, i.e. ;site-bound'. 5. I is important and the EDTA effect at low I is reversible if Na(+) is added immediately after the EDTA: this Na(+) reversibility is lost on standing at 0 degrees . It is suggested that changes in the tertiary structure may be associated with this loss of reversibility. 6. Thermal-denaturation studies show that there is no loss of secondary structure associated with these changes: change in the optical-rotatory-dispersion spectrum in the region of the Cotton effect may be associated with this change in tertiary structure.     

Purification of Escherichia coli amplifiable plasmids by high-salt Sepharose chromatography

This new method allows an easy and rapid purification of amplifiable Escherichia coli plasmids such as pBR 322 without the use of cesium chloride centrifugation. After gentle lysis, centrifugation, and phenol extraction, the material is reextracted with acid phenol to remove the bacterial DNA. The high-molecular-weight ribosomal RNA is removed by precipitation with 2 m ammonium sulfate and the tRNA by passage through a small column of Sepharose CL 4B in the presence of 2 m ammonium sulfate.     

Studies on the interaction of homologues of spermine with deoxyribonucleic acid and with bacterial protoplasts

Four homologues of the naturally occurring polyamine spermine, of the type H(2)N.[CH(2)](3).NH.[CH(2)] (n).NH.[CH(2)](3).NH(2) where n=2, 3, 5 and 6, have been synthesized. Their ability to stabilize Escherichia coli protoplasts against osmotic lysis was compared with that of spermine. All homologues were approximately as effective as spermine. The effect of low concentrations of the homologues on the T(m) of calf thymus DNA and of Aerobacter aerogenes DNA in 0.03m-sodium chloride-1mm-potassium dimethylglutarate buffer, pH6.2, was tested. The increase in T(m) for a given concentration of amine was found to be n=5>n=4 and n=6> n=3>n=2. When calf thymus DNA in 0.15m-sodium chloride-15mm-sodium citrate was used spermine gave the highest increase in T(m). It is concluded that the stabilization of E. coli protoplasts by tetra-amines is a non-specific effect independent of chain length, whereas the elevation of T(m) of DNA is a more specific effect which depends on chain length.     

Photoperiodically induced variation in testicular RNA, DNA and protein content in hamsters and ground squirrels.

1. The total testicular content of RNA, DNA and protein was found to decrease sharply in hamsters with shortened photoperiod and in ground squirrels during the spring breeding season. 2. RNA and DNA per g testes were found to increase in both animals, while protein per g testes remained fairly stable. 3. Cell-free protein synthesis by testicular PMS during testicular regression remained constant when expressed per mg of testicular RNA, but decreased 75% when expressed per testes. 4. These findings suggest that decreases in testicular protein synthesis are due to a decrease in RNA content and not to alteration of the translational activity of the RNA.     

The use of DNA-cellulose for analyzing histone-DNA interactions. Discovery of nucleosome-like histone binding to single-stranded DNA.

In this report, we introduce the use of DNA-cellulose chromatography for evaluating the strength of binding of histones to DNA under a variety of conditions. We have found that histones added directly to DNA-cellulose at physiological salt concentrations bind relatively weakly, with all histones eluting together at about 0.5 M NaCl when a salt gradient is applied. However, much tighter binding of the four nucleosomal histones to DNA-cellulose is obtained if gradual histone-DNA reconstitution conditions are used. In this case, the binding of histones H2A, H2B, H3, and H4 to DNA-cellulose closely resembles their binding to native chromatin. The nativeness of the binding is indicated both by the distinctive sodium chloride elution profile of these histones from DNA-cellulose and by their relative resistance to trypsin digestion when DNA-bound. The binding to DNA-cellulose of histones H2A, H2B, H3, and H4, which have had the first 20 to 30 amino acid residues removed from their NH2 termini, is indistinguishable from the binding to DNA-cellulose of the same intact histones, as judged by their salt elution profile. Thus, even though the NH2 termini contain 40 to 50% of the positively charged amino acid residues (thought to interact with the DNA backbone), a major contribution to the DNA binding comes from the remainder of the histone molecule. Finally, we have discovered that histones can form a "nucleosome-like" complex on single-stranded DNA. The same complex does not appear to form on RNA. Histones H3 and H4 play a predominant role in organizing this histone complex on single-stranded DNA, as they do on double-stranded DNA in normal nucleosomes. We suggest that, in the cell nucleus, nucleosomal structures may form transiently on single strands of DNA, as DNA and RNA polymerases traverse DNA packaged by histones.