A study of DNA denaturation in the ultracentrifuge

The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable molecules melt early and are found at heavier densities in the melting region. An idealized ultracentrifuge melting transition is described. The melting transition of singly nicked PM-2 DNA resembles the idealized curve. The DNA profile is a Gaussian band at all points in the melt. DNA's from mouse, D. Melanogaster, M. lysodeikticus, T4, and T7 also show equilibrium bands at partially denatured densities, some of which are highly asymmetric. Simple sequence satellite DNA shows an all-or-none transition with no equilibrium bands at partially denatured densities. The temperature at which a DNA denatures is an increasing function of the (G + C) content of the DNA. The T_m does not show a molecular-weight dependence in the range 1.2 × 10⁶–1.5 × 10⁷ daltons (single strand) for mouse, M. lysodeikticus, or T4 DNA. The mouse DNA partially denatured bands do not change shape as a function of molecular weight. The T4 DNA intermediate band develops a late-melting tail at low molecular weight. M. lysodeikticus DNA bands at partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components at each point in the melting transition.     

Characterisation of DNA from condensed and dispersed human chromatin

Condensed and dispersed chromatin fractions were isolated from human placental nuclei. The DNA of each fraction was purified and characterised by isopycnic centrifugation, thermal fractionation on hydroxylapatite (HAP) and sequence complexity studies. The DNAs had identical buoyant densities in neutral CsCl (1.698 g/cm³) and similar melting profiles on HAP. Analytical ultracentrifugation in Ag⁺-Cs₂SO₄, however, showed that satellite DNAs were present in the condensed fraction DNA (DNA_C) but were not visible in the dispersed fraction DNA (DNA_D). In addition, DNA_C was found to be enriched in highly reiterated sequences (20% reassociated by C₀t 10^?3) which can be correlated with the presence of satellite DNAs, whereas DNA_D contained only 3% of these fast reassociating sequences. In contrast DNA_D contained 30% intermediate sequences (reassociating between C₀t 10^?3 and C₀t 100) which represent only 10% of DNA_C. The reassociated highly repeated sequences of DNA_C showed the presence of two components in both CsCl density gradients and HAP thermal elution studies. This suggests that either there are sequence relationships resulting in partial mismatching between the different highly repeated DNA sequences in this fraction, or that highly repeated sequences are associated with less repetitious DNA. The results are discussed in terms of possible differences in genetic activity between the chromatin fractions.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

Study of ribosomal deoxyribonucleic acid of sea urchin

The structure of ribosomal DNA (rDNA) satellite of sea urchin (Lytechinus variegatus) sperm has been re-examined after purification by two widely used methods. Saturation hybridization experiments indicate that about 28–32% of the rDNA contain sequences complementary to rRNA. Results of hyperchromic spectral analysis reveal that the rDNA melts as two distinct but unequal components. The early transition presumably corresponds to the melting of most of the transcribed part and the late transition is suggestive of the melting of a G+C-rich segment of the rDNA, which may be a non-transcribed spacer.Abbreviations F_AT fraction of all A+T pairs denatured - F_GC fraction of all G+C pairs denatured - SSC standard saline citrate - rDNA template for ribosomal RNA This work is a part of the author's Ph.D. thesis (U.N.C., Chapel Hill).     

Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis: renaturation and sedimentation studies

The renaturation kinetics of mitochondrial DNA from the yeast Saccharomyces carlsbergensis have been studied at different temperatures and molecular weights. At renaturation temperatures 25 deg. C below the mean denaturation temperature (T_m) in 1 M-sodium chloride the renaturation rate constant is found to decrease with increasing molecular weight of the reacting strands. This unusual molecular weight dependency gradually disappears with an increase in the renaturation temperature. At a temperature 10 deg. C below the melting point, the rate constant shows the normally expected increase with the square root of the molecular weight. From the renaturation data at this temperature, the molecular weight of the mitochondrial genome is estimated to be about 5·0 × 10⁷. The same size of genome was found from renaturation at low molecular weight and 25 deg. C below the T_m.The sedimentation properties of denatured mitochondrial DNA at pH values 7·0 to 12·5 were used to study the conformation of this DNA in 1 M-sodium chloride. The results obtained support the conclusion from the renaturation studies: that the pieces of denatured mitochondrial DNA with a molecular weight above 2 × 10⁵ to 3 × 10⁵, in 1 M-sodium chloride at 25 deg. C below the mean denaturation temperature are not fully extended random coils. Presumably, interaction between adenine and thymine-rich sequences, which are clustered at certain distances within the molecules, is the molecular basis for these observations.     

The termini of bacteriophage T7 DNA

The termini of Escherichia coli phage T7 DNA have been labeled with ³²P by the polynucleotide kinase reaction. The DNA was fragmented, denatured, and annealed to denatured T7 DNA embedded in agar; elution was measured as a function of temperature. The terminal fragments were eluted from the gel at temperatures well below that of the bulk of the DNA, suggesting that these regions have a very high adenine-plus-thymine content. However, when DNA doubly labeled throughout at random by growth of the phage in [³H]thymidine and ³²PO₄, was denatured, annealed to the gel, and eluted as a function of temperature, the material eluting from the gel in this low-temperature range was about 50% adenine and thymine. Hence the melting behavior of the terminal fragments is not a result of a high adenine plus thymine content. By electrophoretic analysis of exonucleolytic digests of the T7 DNA it was shown that no unusual bases were present. It is suggested that the low thermal stability of the annealed terminal fragments is a consequence of the high guanine·cytosine regions being unavailable for hybridization, possibly because they are involved in intra-strand hydrogen bonding.     

A study of sequence homologies in four satellite DNAs of man.

Satellites I, II, III and IV (Corneo et al., 1968,1970,1971) have been purified from human male placental DNA. The sequences present in these four DNA components have been characterized by analytical buoyant density, thermal denaturation, DNA reassociation, DNA hybridization and gel electrophoresis coupled with hybridization following either HaeIII or EcoRI restriction endonuclease digestion. Satellites III and IV were found to be virtually indistinguishable by a variety of criteria. Cross-satellite reassociation showed that 40% of the molecules present in satellite III contain sequences that are homologous to 10% of the molecules of either satellite I or satellite II. Reassociated satellite I melts as a single component, as do the hybrid duplexes between satellite I and satellite III. In contrast, reassociated satellites II, III and IV, and the hybrid duplexes formed between satellites II and III and between satellites II and IV, melt as two distinct components with different thermal stabilities.Digestion of satellite III with HaeIII gives rise to a series of fragments whose sizes are 2, 3, 4, 5, 6, 7, 8 and 11 times the size of the smallest 0.17 × 10³ basepair fragment, in addition to a 3.4 × 10³ base-pair male-specific fragment (Cooke, 1976) and high molecular weight material. The sequences contained in the fragments of the HaeIII ladder are diverged from each other as well as being non-homologous with those of the 3.4 × 10³ base-pair and high molecular weight fragments. The latter contain EcoRI recognition sites. Satellite II has a similar pattern of fragments to satellite III following digestion with HaeIII, although it can be distinguished from satellite III on the basis of the products of EcoRI digestion. Satellite I contains neither HaeIII nor EcoRI recognition sites. The cross-satellite homologies of the sequences present in fragments of differing sizes produced by restriction enzyme digestion have also been studied.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma

DNA isolated from purified nuclei of Polytoma obtusum has a buoyant density of 1.711 g/ml in CsCl, a Tm of 91.3° C in SSC, and a G + C content of 52.5% as determined by base composition analysis. Thermal dissociation and reassociation studies indicated that this nuclear DNA contains a considerable amount of heterogeneity. Under appropriate reannealing conditions for denatured DNA, about 15% of the DNA reannealed to form a satellite peak at a density of 1.711 g/ml within one hour. Native DNA fractions of different average buoyant densities, ranging from 1.723 to 1.708 g/ml were also obtained in a preparative CsCl gradient, indicating the presence of intermolecular heterogeneity at a molecular size of 8.5×10⁶ daltons. The nuclear DNA reassociated as three distinct classes. The very fast species constituted about 20 % of the total hyperchromicity, the class of intermediate rate comprised roughly 10% of the nuclear DNA, while the remaining 70% consisted of unique sequences. The haploid genome set was estimated by renaturation kinetics studies to contain 5.0×10¹⁰ daltons of DNA or 7.5×10⁷ nucleotide pairs. The analytical complexity of the total nuclear genome was found to be 9.35×10¹⁰ daltons, thus indicating that vegetative cells of P. obtusum are diploid.     

Fractionation of calf thymus DNA based on its interaction with homologeous f1 histone. Melting curves of the obtained fractions

Calf thymus DNA fractions were obtained by precipitation with the homologeous f₁ histone and their melting curves were investigated. An increase of the melting temperatures of DNA remaining in the supernatants was observed. Within the range of 5–50 % of the DNA precipitated the melting temperatures and the melting intervals of DNA in the sediment remained constant. The obtained values /38 mole % GC, ΔT_m = 7.0°C/ suggest that DNA found in the precipitates corresponds to the main calf thymus DNA. Despite its heterogeneity this group of DNA molecules does not undergo fractionation using f₁ histone. We assume that the molecules of the main DNA all contain specific areas to which the f₁ histone attaches in our experimental conditions. The main DNA molecules, regardless of their base composition, seem to contain these specific areas in amounts causing equal precipitation probability. They seem to differ in this respect from some GC rich fractions, possibly those of satellite DNA.     

Spectral Analysis of Naturally Occurring Methylxanthines (Theophylline,Theobromine and Caffeine) Binding with DNA

Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR) spectroscopic methods, and especially monitored their binding affinity in the presence of Mg²⁺ and during helix-coil transitions of DNA by temperature (T_m) or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophylline = 3.5×10³ M⁻¹, DNA-theobromine = 1.1×10³ M⁻¹, and DNA-Caffeine = 3.8×10³ M⁻¹. On the other hand T_m/pH melting profiles showed 24–35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C) and phosphate group through hydrogen bond (H-bond) interaction. In the presence of Mg²⁺, methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg²⁺. The spectral analyses indicated the order of binding affinity as “caffeine≥theophylline>theobromine” to the native double helical DNA, and “theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions.     

Restriction Enzyme Digests of Rapidly Renaturing Fragments of Vaccinia Virus DNA

Vaccinia virus DNA fragments that have been denatured by alkali and then neutralized contain a fraction that rapidly reforms duplex structures. The fraction is enriched by fractionating on hydroxyapatite columns and serves as a substrate for digestion by two restriction endonucleases isolated from Hemophilus parainfluenzae, Hpa I and HPa II. The patterns obtained by gel electrophoresis of the digested fragments show the presence of three major bands after Hpa I digestion and four major bands after Hpa II digestion. The DNA that is isolated from some of these bands quickly reforms duplex regions after alkaline denaturation. The size of the DNA segments in the major bands has been estimated to be in the range of 0.44 × 10⁶ to 3.2 × 10⁶ daltons. The fragments which rapidly reform duplex chains after denaturation are sensitive to single-strand-specific nucleases. These results are consistent with a model of vaccinia virus DNA which has a covalent link connecting complementary chains.     

Characterization of the most rapidly renaturing sequences in mouse main-band DNA

The most rapidly renaturing sequences in the main-band DNA of Mus musculus, isolated on hydroxyapatite, are found to consist of two discrete families: a presumed “foldback” DNA fraction and a fraction renaturing bimolecularly. The latter family, which we call “main-band hydroxyapatite-isolated rapidly renaturing DNA”, has a kinetic complexity about an order of magnitude greater than that of mouse satellite DNA. It shows about twice as much mismatching as renatured mouse satellite, as judged by its thermal denaturation curve. In situ hybridization localizes the sequences to all chromosomes in the mouse karyotype, and to at least several regions of each chromosome. The in situ result and solution hybridization studies eliminate the possibility that the main-band rapidly renaturing DNA is composed of mouse satellite sequences attached to sequences of higher buoyant density. Nuelease S₁ digestion experiments disclose that even at low molecular weight there are unrenatured “tails” attached to the rapidly renaturing sequences. When the main-band DNA fragment size is increased the amount of rapidly renaturing sequences remains constant, but the amount of attached tails of unrenatured DNA increases as judged by S₁ nuclease digestibility, hyperchromicity and buoyant density. It is concluded that at least 5% of the mouse genome is composed of segments of the rapidly renaturing sequences averaging about 1500 base pairs, alternating with segments of more complex DNA averaging about 2200 base pairs. This interspersion of sequences is compared to that found in several other organisms. The properties of the foldback DNA are similarly investigated as a function of DNA fragment size.     

DNA Polymerase Activity Associated with the Membrane Fraction of E. coli

Spheroplasts were disrupted with 0.2% Brij 58 and the separation of intact cells, spheroplasts, disrupted spheroplasts, fragmented membrane, and supernatant was performed on a linear 40~55% sucrose gradient. About half an amount of nucleic acid components was distributed in disrupted spheroplast fractions, while only a small amount of protein components was found in these fractions.

DNA polymerase in the fragmented membrane fraction incorporated ³H-TTP more rapidly than that in the supernatant fraction for the first 5 to 6 min, and then the incorporation rate decreased, while DNA polymerase in the supernatant fraction incorporated ³H-TTP linearly up to 20 min when native DNA was used as a primer. The former required native DNA as a primer and showed little activity towards denatured DNA, while the latter incorporated ³H-TTP at a similar rate to both the primer DNA’s.

DNA polymerase of the fragmented membrane fraction synthesized various sizes of DNA from short to a size of primer when native DNA was used as a primer, while when denatured DNA was used, products were only short. DNA polymerase of the supernatant fraction synthesized various sizes of DNA when both native and denatured DNA’s were used as primers.     

Renaturation of denatured DNA in NaI gradients

We show that denatured DNA from Tetrahymena mitochondria or phage T₇, partially renatures during centrifugation in NaI equilibrium density gradients. This makes these gradients unsuitable for the analysis of single-stranded complementary nucleic acids, especially if their complexity and mole percent G+C are low.     

Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow

1. Rats were injected with [U-¹⁴C]glucose and the content of ¹⁴C in proteins and lipids of the cerebral P₁ (`nuclear'), P<sub>2</sub> (`mitochondrial'), P₃ (`microsomal') and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P<sub>2</sub>) were subfractionated on continuous sucrose gradients (0·32–1·8m-sucrose) and the <sup>14</sup>C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40–50% of the <sup>14</sup>C assimilated by brain proteins was found in the P<sub>2</sub> (`mitochondrial') fraction. About 68–70% of the ¹⁴C assimilated by brain lipids was also recovered from the lipids of the P₂ fraction. 4. Between 22 and 93hr. after injection of [U-¹⁴C]glucose both the amount of ¹⁴C in the protein of the P₂ (`mitochondrial') fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P₁, P₂ and P₃) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P₂) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U-¹⁴C]glucose. The amount of ¹⁴C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of ¹⁴C in the lipids of all subcellular particulate fractions remained unchanged during the period 22–93hr. after injection of [U-¹⁴C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.     

Identification and mass analysis of human fibrinogen molecules and their domains by scanning transmission electron microscopy

The domainal substructure and molecular conformation of human fibrinogen have been investigated by evaluating scanning transmission electron microscopic images of freeze-dried or negatively contrasted native fibrinogen (fractions I-4 and I-9), glutaraldehyde-treated fibrinogen, or plasmic core fragments D₁ and E₂. Although some unstained freeze-dried native or glutaraldehyde-treated fibrinogen molecules were relatively compact and even occasionally spheroidal, typical images were elongated symmetrical tridomainal structures 460 Å ± 20 Å in length; frequently they were bent into a variety of elongated though non-linear arrangements. Their identification as monomolecular forms of fibrinogen by scanning transmission electron microscopic mass measurements resolved uncertainties relating to the identity of such objects as single molecules. The central domains of fraction I-4 molecules had a greater mass than those of fraction I-9 (1.01 × 10⁵M_rversus 7.5 × 10 M_r, respectively). This difference accounted for the observed mass difference between fraction I-4 and fraction I-9 molecules (i.e. 3.27 × 10⁵M_rversus 2.97 × 10⁵M_r, respectively) and suggested that the COOH-terminal region of the Aα chain (major portions of which are always absent from fraction I-9 molecules) is situated within the mass integration radius for the central domain. When the COOH-terminal region of the Aα chain was present it appeared in negative stain as a thread-like structure originating between the middle and outer domains and extending toward the central domain, sometimes appearing to wind around the long axis.The outer domains of negatively stained molecules resembled negatively stained images of fragment D₁ and could frequently be resolved into at least two discrete subdomains, forming an oblong structure usually canted at an angle of ～120 ° to 150 ° relative to the long axis. Our findings are consistent with prevailing tridomainal structural models of fibrinogen and suggest that these molecules are flexible and may exist in unfolded configurations, or as relatively compact, partially or completely folded forms.     

Role of calcium binding by sarcoplasmic reticulum in the contraction and relaxation of uterine smooth muscle

The binding of calcium by isolated sarcoplasmic reticulum from cow uterus was studied. Sarcoplasmic reticulum was prepared by differential centrifugation. Three fractions were obtained: I, sedimented between 2,500–15,000 x g; II at 40,000 x g; and III, at 150,000 x g. Fraction II was further purified on a sucrose density gradient. All three fractions contained considerable amounts of intrinsic calcium, mostly in fraction I. Calcium binding in the presence of ATP¹ and Mg also was greatest in fraction I, followed by fraction II, with less in fraction III. Without ATP no calcium was taken up. 5 and 10 mM sodium azide partially inhibited calcium binding in fraction I, but not in fraction II, suggesting the presence of some mitochondria or mitochondrial fragments in fraction I. Calcium binding in fraction II was completely inhibited by 3 mM salyrgan; this fraction thus appears to be sarcoplasmic reticulum. ATPase activity was found in all three fractions, highest in fraction II. It is computed that calcium binding in fractions I and II, on the basis of a 50% yield of protein, is sufficient to elicit contraction by supplying calcium to the contractile proteins of the smooth muscle cell and to regulate relaxation and contraction.