Structure and physico-chemical properties of bacteriophage G: III. A homogeneous DNA of molecular weight 5 × 108

The physico-chemical properties of the DNA released from bacteriophage G (active on Bacillus megatherium) are described. Phage G, an unusually large bacteriophage, has a nucleic acid content of 4 to 6 × 10⁸ daltons.Sedimentation velocity analysis at low angular speed and examination by electron microscopy, indicate that a single DNA molecule, sedimenting with s_{20, w}⁰ = 125 ± 1.5 S and at least 200 ± 20 μm long, is released upon thermal or osmotic shock. Melting temperature data and chromatographic analysis indicate a mean base composition of 70% A + T. CsCl and Cs₂SO₄ buoyant density data, circular dichroism spectra and sensitivity to specific nucleases indicate that phage G DNA is similar to the DNAs from T-even phages and is more glucosylated than phage T6 DNA. Direct glucose determination indicates a 185% molar ratio of glucose to cytosine. Linear density extrapolated from literature data and contour length measurement yield a lower limit for the molecular weight of phage G DNA of 4.9 × 10⁸. Comparison of this value with the s_20,w⁰ measured with the analytical ultracentrifuge seems to confirm the validity of the empirical relationship proposed by Freifelder (1970), between s_{20, w}⁰ and molecular weight, over a larger range than that previously known. A possible systematic error in defect in length determination, however, prevents a discrimination between this and other empirical formulae proposed by various authors, which predict a molecular weight that is 20 to 25% higher.     

The flexibility of low molecular weight double-stranded dna as a function of length: II. Light scattering measurements and the estimation of persistence lengths from light scattering,sedimentation and viscosity

In the preceding paper are described the isolation and physical characterization of seven narrowly disperse fractions of calf thymus DNA in the molecular weight range 0.3 to 1.3 × 10⁶ daltons. Herein, we have determined by light scattering the molecular weights and root mean square radii of these fractions in a solvent comprising 0.2 M NaCl, 2 mM EDTA, 2m MNa-PO₄, pH 7. Measurements were made in a modified Wippler—Scheibling photometer to a 20° lower limit of scattering angle on solutions rendered virtually dust-free by procedures described. The optical aniso tropics of the DNA fractions were measured permitting the experimental molecular weights and root mean square radii to be corrected to their true values. From these values, with appropriate polydispersity corrections, we calculate a Kratky—Porod persistence length, a, of 54.0 ± 5.6 nm which is invariant over the molecular weight range examined. From the sedimentation coefficients (preceding paper) and the theory of Yamakawa and Fujii, we calculate a to be 66 nm, a value found to apply equally well to several DNA samples of various origins whose sedimentation rates are known in the molecular weight range from about 4 × 10⁴ to 10⁸ daltons. Similarly, from the intrinsic viscosities and the theory of Yamakawa and Fujii, we calculate a to be 59 nm, which again adequately applies to a number of DNA samples whose viscosities have been measured by other workers in the molecular weight range 3 × 10⁵ to 10⁸ daltons. The Flory—Mandelkern parameter, β, was found to vary with molecular weight in the manner predicted by the theory of Yamakawa and Fujii. The average value of a from the three sets of measurements is 60 ± 6 nm, which we believe applies to double-stranded DNA molecules, independent of chain length, over the whole range of molecular weights for which reliable data exist.     

DNA-Protein Complex in Circular DNA from Phage ϕ29

THE DNA of the B. subtilis phage ?29 has been described as unpermuted linear duplex molecules¹ of molecular weight 11 × 10⁶, but the formation of circular molecules has also been indicated, suggesting the existence of cohesive ends^1,2.     

DNA of Myxococcus bacteriophage MX-1: Macromolecular properties and restriction fragments

1. Bacteriophage MX-1 is a virulent DNA phage whose hosts include strains of Myxococcus xanthus, M. fulvus and M. virescens. DNA was extracted from purified phage preparations. The molecular weight of phage DNA was measured by sedimentation-velocity and by rate-zonal ultracentrifugation. The apparent molecular weight was found to vary for reasons discussed in the text. From ratezonal ultracentrifugation, using calibrated sucrose gradients, the molecular weight was calculated to be 149 (± 22)×10⁶ daltons. The base composition of the DNA was estimated by different methods and was found to be 50–52% (G+C). The DNA demonstrated an anomalous thermal denaturation profile in dilute buffer. Denatured DNA was fractionated by ion-exchange chromatography and by buoyant-density centrifugation. No significant strand separation was obtained and it was concluded that overall base compositions of the two strands are very similar.
2. DNA from bacteriophage MX-1 was hydrolysed with restriction endonucleases R. EcoRI, R. EcoRII and R. HindIII. The restriction fragments were catalogued and their apparent molecular weights calculated from electrophoresis gels calibrated with fragments from the DNA of coliphage λ. From the total fragments obtained with nuclease R. EcoRI, the minimum apparent molecular weight of MX-1 DNA was found to be 130×10⁶ daltons.

     

The flexibility of low molecular weight double-stranded dna as a function of length: I. Isolation and physical characterization of seven fractions

Sonicated calf thymus DNA was fractionated by rate zonal centrifugation into seven fractions with weight average molecular weights ranging from 0.28 to 1.3 × 10⁶ daltons, as determined by sedimentation equilibrium and light scattering measurements (the latter are described in the accompanying paper). Electron microscopy and sedimentation equilibrium analysis revealed these fractions to be narrowly disperse with M_w/M_n ratios averaging about 1.06. Intrinsic viscosities and sedimentation rates were measured and found to vary linearly with molecular weight in double-logarithmic plots in fair agreement with previously published functions relating these parameters for low molecular weight DNA. The average value for β from the Mandelkern— Flory equation was 2.59 × l0⁶, also agreeing with reported estimates of this parameter for short DNA. These data will be used in the second paper of this series to calculate the persistence length of the DNA fragments in each of the seven fractions by light scattering and hydrodynamic theories for the Kratky—Porod worm-like coil.     

Structural polypeptides and products of late genes of bacteriophage Mu: Characterization and functional aspects

This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion.In vitro reconstitution of infectious phage particles has shown that four genes (E, D, I, J) control the formation of phage heads and that a cluster of eight genes (K, L, M, N, P, Q, R, S) controls the formation of phage tails.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10³M_r); H (64 × 10³M_r); F (54 × 10³M_r); G (16 × 10³M_r); L (55 × 10³M_r); N (60 × 10³M_r); P (43 × 10³M_r) and S (56 × 10³M_r). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V, W or R (41.5 × 10³M_r, and 45 × 10³M_r); U (20.5 × 10³M_r) and of genes located in the β region (24 × 10³M_r (gpgin) and 37 × 10³M_r (possibly gpmom)).Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10³, a second head polypeptide of 54 × 10³ (gpF) and two major tail polypeptides with molecular weights of 55 × 10³ and 12.5 × 10³ (gpL and gpY, respectively). In addition, there are five minor components of the tail (including gpN, gpS and gpU) and approximately seven minor components of the head structure of the virion (including gpH).     

Characterization of the deoxyribonuclease determined by lambda reverse as exonuclease VIII of Escherichia coli.

Gottesman et al. (1974) detected a new DNAase in Escherichia coli infected with λ reverse, a recombination-proficient substitution mutant of phage λ which is deleted for the λ recombination genes. We have purified this enzyme, using the procedure developed for the purification of exonuclease VIII (Kushner et al., 1974), a DNAase produced by E. coli K-12 strains carrying sbcA^? mutations. The λ reverse exonuclease (Exoλrev) is identical to exonuclease VIII by several criteria. The two enzymes elute at similar salt concentrations from DEAE-cellulose and DNA-cellulose; sediment at the same velocity in glycerol gradients, corresponding to a molecular weight of about 1.4 × 10⁵; migrate at the same R_F in sodium dodecyl sulfate/polyacrylamide gels, indicating a polypeptide molecular weight of 1.4 × 10⁵; exhibit maximum activity at 20 mm-Mg²⁺ and pH 8 to 9; and are much more active on double-stranded DNA than on heat-denatured DNA. Both enzymes are rendered sedimentable by antiserum against Exoλrev. This evidence supports the hypothesis that the non-λ DNA substitution in λ reverse includes recE, the structural gene for exonuclease VIII.     

The influence of rotor speed on the sedimentation behavior in sucrose gradients of high molecular weight DNA's

Anomalous sedimentation behavior has been observed for high molecular weight duplex DNA's in sucrose gradients. The sedimentation rate of DNA's having molecular weights of 10⁸ or higher is influenced by high centrifugal fields. The change in the sucrose sedimentation coefficient due to this effect, S^RPM_suc-S⁰_suc, is equal to 1 × 10^?48M^3.65( The anomalous behavior is not influenced by DNA concentration at sufficiently low concentrations. Because of the smallness of the coefficient this effect has not been previously detected for DNA's the size of T2 or smaller at rotor speeds below 40000 RPM. For example, the relative sedimentation coefficient of T2 DNA at 65 000 RPM is only 9% less than at 10000 RPM. However, the sedimentation profile of heterogeneous high molecular weight [(100 – 350) × 10⁶] E. coli DNA is severely altered even at moderate rotor speeds (37000 RPM). Therefore, it seems advisable to use low rotor speeds when sedimenting high molecular weight DNA's.     

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.     

Homology of plasmids in strains of unicellular Cyanobacteria.

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 10⁶ and 32.7 × 10⁶. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 10⁶ and 24 × 10⁶. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 10⁶ and 2.0 × 10⁶, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.     

Physical characterization of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

A generalized transducing bacteriophage of Myxococcus xanthus has been examined. The phage particle consists of an isometric head and a contractile tail. The genome of the phage is a linear DNA molecule of molecular weight 39 ± 2.1 × 10⁶, which contains the normal DNA bases 70% of which are guanosine + cytosine. No overall heterogeneity of base composition is present. The DNA does not carry easily detectable cohesive ends nor is it cyclically permuted. It does contain a large and somewhat variable terminal redundancy. Heating phage particles in the presence of EDTA causes tail sheath contraction and ejection of DNA, some of which remains attached to the tail. Digestion of tail-bound DNA with restriction enzymes shows that the phage tail can be attached to either end of the DNA. Thus the DNA probably contains recognition sites for the packaging of its DNA at both ends. These results suggest possible mechanisms for the genesis of transducing particles by phage MX4.     

Mammalian Metaphase Chromosomes with High Molecular Weight DNA isolated at <Emphasis Type="Italic">p</Emphasis>H 10.5

MAMMALIAN metaphase chromosomes may be isolated either at an acid pH^1–4 or at nearly neutral pH^5–7. The only exception is the method of Corry and Cole⁸, performed at pH 9.5. We used alkaline sucrose velocity gradients to determine the size distributions of DNA (molecular weight) in metaphase chromosomes in an attempt to understand their arrangement. Initial experiments yielded 1 × 10⁶ molecular weight DNA (single stranded) pieces regardless of chromosome size. When metaphase cells are lysed directly on the gradient a high molecular weight (1 × 10⁸ for single stranded) is obtained. We therefore examined a large number of chromosome isolation procedures and cytological conditions to determine their effects on the molecular weight of the DNA.     

Dynamic light scattering of the bacteriophage T4B: determination of translational diffusion coefficients and centres of rotation

Using dynamic light scattering, the translational diffusion coefficient (D_T) and the distance between the hydrodynamic centre and the centre of the head (r₀) of the bacteriophage T4B have been determined. For a particle with retracted tail fibres we found D_T20.w =2.88 (2.88 ± 0.02) × 10^?8cm²s^?1 and r₀ = 52 ± 1 nm. For a phage with fully extended tail fibres D_T20w = (.210 ± 0.02) × 10^?8cm²s^?1 and r₀ = 112 ± nm. These data were obtained by interpreting the correlation function using a theory which takes into account the influence of the lollipop shape of the phage. In the literature this influence has not been taken into account, which has led to erroneous values of diffusion coefficients for T4B and other phages. The sedimentation coefficient of T4B phage is 1040 ± 5 S (fibres retracted) or 829 ± 4 S (fibres extended). With the above mentioned diffusion coefficients, these values correspond to a molecular weight of 236 × 10⁶ ± 3 × 10⁶. Finally, the theory used in this study is applied to other bacterial viruses, to correct reported values of the translational diffusion coefficients and of the corresponding molecular weights of these viruses.     

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.     

Dielectric relaxation of DNA solutions. II

Real and imaganiry parts of complex dielectric constant of dilute solutions of DNA in 10^?3M NaCl with molecular weight ranging from 0.4 × 10⁶ to 4 × 10⁶ were measured at frequencies from 0.2 Hz to 30 kHz. Dielectric increments Δε were obtained from Cole-Cole plots and relaxation times τ_D from the loss maximum frequency. The τ_D of all samples agrees well with twice of the maximum viscoelastic relexation time in the Zimm theory, indicating that the low-frequency dielectric relaxiation should be ascribed to be the rotation of DNA. The rms dipole moment, which was obtained from Δε, agree well with that calculated from the counterion fluctuation theory. The dielectric increment was found to be greatly depressed in MgCl₂, which is resonably interpreted in terms of a strong binding of Mg⁺⁺ ions with DNA.     

The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10⁶ (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10⁶. On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10⁶-dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10⁶, present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10⁶, present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10⁶, independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo.     

Analysis of low angle light scattering results from T7 DNA

Selected light scattering data, obtained in earlier studies on T7 DNA in 0.195 M Na⁺, are analyzed by comparison with calculations from the theory of wormlike coils, both with and without excluded volume effects. The results confirm the conclusion from an earlier criticism, that linear extrapolations of data from the 10° to 20° angular range give incorrect values for the limiting molecular weight, M_T, and for the limiting root-mean-square radius, R_T. Further, it is shown that the excluded volume parameter, ?, must be used to provide a proper fit of calculated curves to experimental data. The revised analysis gives the following parameters for T7 DNA: M_T = 25.5 × 10⁶ ;R_T= 587 nm; ? = 0.08; and the statistical segment length, 1/λ = 120 nm. These parameters agree well with other values in the literature. The method of analysis, therefore, provides reliable results from light scattering data on high-molecular-weight, native DNA.     

Occurrence in vivo of "hidden breaks" at specific sites of 26 S ribosomal RNA of Musca carnaria.

A fragmentation process occurs in 26 S ribosomal RNA of mature cytoplasmic ribosomes of Musca carnaria. It consists of the sequential appearance of three “hidden breaks” that fragment 26 S rRNA (M_r = 1.42 × 10⁶) into four pieces with approximate molecular weights of 0.68 × 10⁶, 0.35 × 10⁶, 0.29 × 10⁶ and 0.096 × 10⁶, respectively. This fragmentation was not observed in 17 S rRNA (M_r = 0.74 × 10⁶).Extremely mild treatment of newly assembled ribosomes with pancreatic RNAase reproduces the 26 S rRNA fragmentation phenomenon in vitro in the same way as it occurs in vivo.This evidence is discussed in relation to the secondary structure of 26 S rRNA and its binding with specific ribosomal proteins.     

A circular channel crucible oscillating viscometer: Detection of DNA damage induced in vivo by exceedingly small doses of dimethylnitrosamine

A new oscillating crucible viscometer, having a U-shaped circular channel, is described. The damping coefficient δ is lowered by an increase of the viscosity η. The instrument described here allows the solution to come in contact with inert plastic only. At all steps of its preparation and during viscosity measurements, giant DNA from rat liver nuclei was maintained at shear stresses around 10^?4 dynes cm^?2. Viscosity was studied as a function of surface tension, DNA concentration and shear stress. It was found that under our experimental conditions it was possible to obtain meaningful values for reduced viscosity, η_red, practically identical to intrinsic viscosity [η]. Rat liver nuclei are incubated in an alkaline lysing solution (pH 12.5; 22 °C): they are lysed immediately and the released DNA starts to uncoil. The viscosity of solutions of this giant DNA increases very slowly with time, reaching a maximum only after about ten hours. The process was accelerated by single-stranded breaks arising from methylation of DNA in vivo with dimethylnitrosamine. It was found that the time of DNA disentanglement was sensitive to an exceedingly small number of breaks. We think that we were able to measure molecular weights around the length of the single strand of an average chromosome (M_n 5 × 10¹⁰). An empirical relation between molecular weight and reduced viscosity after complete disentanglement was also established, as a linear log-log plot, covering a molecular weight range between 10⁸ and 2.5 × 10¹⁰. It is suggested that the viscosimetric evaluation of DNA disentanglement is probably the most sensitive method for studying DNA damage induced “in vivo” by chemical carcinogens.