A quantitative test of Zimm's model for the rotor-speed-department sedimentation of linear DNA molecules

In 1974, Zimm described a theory which predicts that the sedimentation coefficient of high-molecular-weight DNA will decrease as the rotor speed of measurement increases. In 1979, this theory was revised, and the new formula predicts speed-dependence effects that are substantially smaller than the predictions of the original version. This report describes the results of subjecting both the original and the revised versions of the theory to quantitative tests using a well-defined sucrose-gradient system and a DNA of known molecular weight (T4c DNA). T4c bacteriophage is a mutant, whose DNA contains the unmodified base cytosine, instead of the glucosylated hydroxymethylcytosine characteristic of the T-even bacteriophages, and has a molecular weight of 115 ± 3 × 10⁶. The DNA of the wild-type phage (T4D⁺) was also used in some experiments. In addition to the quantitative tests, the experiments test for an effect first observed by Rubenstein and Leighton, which showed that the sedimentation coefficient measured for T2 DNA depended on the composition of the centrifuge tube used for the measurement (tube composition effect). It can be inferred from this observation that an interaction occurs between particle and tube wall during sedimentation, and this leads to a reduction in sedimentation velocity independent of the reduction in S described by Zimm's theory. The results show that in the range of 25,000–50,000 rpm, the original but theoretically incorrect form of the theory quite accurately describes the sedimentation behavior of both T4c and T4D⁺ DNA, although T4D⁺ was a special case in some respects. The revised (corrected) form of the theory predicts much less of a speed-dependence effect than that actually observed. The discrepancy between corrected theory and observation suggests that other factors (perhaps arising from the use of the swinging bucket rotor geometry) are causing the additional observed reduction in S_20,w. However, the experiments show that the tube composition effect does not seem to be one of these.     

Replicative Intermediates of Bacteriophage T7 Deoxyribonucleic Acid

After infection with bacteriophage T7, parental and newly synthesized deoxyribonucleic acid (DNA) exhibit an extremely fast sedimentation rate in neutral sucrose gradients. This fast-sedimenting component (intermediate I) has a sedimentation constant of about 1,500S and contains T7 DNA as determined by DNA-DNA hybridization experiments. Pulse-chase experiments indicate that the fast-sedimenting material is metabolically active and serves as a precursor to the formation of T7 DNA. Intermediate I contains about 2.5 to 7% of the total ³H-labeled protein formed between 3 and 9.5 min after T7 infection. Treatment of intermediate I with Pronase results in the release of the DNA from the complex. At early times after infection, a second intermediate (intermediate II) can be detected which contains both parental and newly synthesized DNA sedimenting slower than intermediate I but 2 to 3 times as fast as mature T7 DNA. Intermediates I and II containing parental DNA are formed after infection of the nonpermissive host with an amber mutant in gene 1, a gene whose expression is necessary for the synthesis of most T7 proteins. The two intermediates are also observed when infection with T7 wild type is carried out in the presence of chloramphenicol.     

Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation

A method is presented for preparing very large DNA from Bacillus subtilis protoplasts. When the DNA is characterized by sedimentation in neutral sucrose gradients, a fast-sedimenting component is found whose sedimentation coefficient varies with centrifuge speed. By use of [³H]thymine label for the DNA and a ¹⁴C-labeled amino acid, it is shown that less than 5% cellular material other than DNA is associated with this component. Irradiation of this DNA in solution with gamma rays forms a slower component, called the “main peak”, whose sedimentation coefficient also depends on centrifuge speed. More irradiation breaks down this main peak into even slower-sedimenting DNA; it is shown that for low doses, double-strand breaks are formed in both the B. subtilis DNA and in bacteriophage T2 DNA at the same rate linear in dose, 0.018 double-strand breaks per kilorad per mass equal to that of T2 DNA.The speed dependence of the DNA sedimenting at the main peak is compared with an approximate theory of the speed dependence of the sedimentation coefficient of linear DNA by B. H. Zimm (unpublished calculations). The comparison suggests that for sufficiently high centripedal acceleration, DNA molecules larger than a critical mass will sediment at much the same velocity. The theory, and data on the break-up of the DNA with gamma rays, are used to estimate that the DNA extracted is at least 13 times the mass of T2 DNA, and possibly larger.In the Appendix, data from the literature are put together with data taken during this work to make plausible the assumption that the usual theory for the sedimentation of DNA molecules, experimentally tested in salt solutions, may also be applied to sucrose solutions. If, in neutral sucrose gradients, the distance sedimented is proportional to a power α of the mass, the best value of α = 0.38.     

Rotor speed dependent sedimentation of circular and linear DNA

The sedimentation rate of large, linear DNA molecules has been shown to be rotor speed dependent (Rubenstein and Leighton, Biophys. Chem. 1 (1974)). In this communication we report the first studies designed to measure the rotor speed effect with a homogenous, linear viral DNA larger than bacteriophage T2 DNA. We also report the first studies using a homogenous, circular episomal DNA of known molecular weight. For this circular DNA a small rotor speed effect, previously unsuspected, was discovered.     

Rotor speed dependent sedimentation of circular and linear DNA.

The sedimentation rate of large, linear DNA molecules has been shown to be rotor speed dependent (Rubenstein and Leighton, Biophys. Chem. 1 (1974)). In this communication we report the first studies designed to measure the rotor speed effect with a homogenous, linear viral DNA larger than bacteriophage T2 DNA. We also report the first studies using a homogenous, circular episomal DNA of known molecular weight. For this circular DNA a small rotor speed effect, previously unsuspected, was discovered.     

Anomalies in sedimentation. I. Stability theory of sedimenting entanglements in the "tight-bending" limit

A simple model is introduced to investigate the stability of a sedimenting entanglement. The sedimenting entanglement is represented by a sedimenting sieve. Solvent can pass through it, but single-chain molecules that flow into it become entangled and their flow decreases or, if permanent entanglements form, ceases entirely. With this model we are able to find the conditions under which the mass of a sedimenting entanglement remains constant, grows or decays to a stable value, grows beyond limit, or decays to the mass of a single chain. The theory is applied to the sedimentation of small concentrations of large chain molecules in solutions of small chain molecules in solutions of small chain molecules for the case in which the entanglements are long-lived. Equations are derived which, (1) give the stable entanglement mass as a function of rotor speed and concentration and, (2) for a given concentration predict the rotor speed at which the entanglement mass grows without limit. Numerical results for small concentrations of T2 DNA sedimenting in solutions of T7 DNA are presented.     

Anomalies in sedimentation. II. Testing the entanglement hypothesis

Zones of T2 DNA were sedimented through uniform solutions of T7 DNA to determine if the smaller DNA molecules would become entangled in the larger. No entanglement could be demonstrated even at high DNA concentrations. It is suggested that molecular entanglement is not responsible for the sudden loss of DNA from solution which occurs in high centrifugal fields. This communication also includes observations on the effects of rotor speed on the sedimentation behavior of DNA in high centrifugal fields, distortion of zone shape at high concentrations, and hydrodynamic interactions between DNA and MS2 bacteriophage particles.     

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.     

Theory and practice of centrifugal elutriation (CE)

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that aselective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5×10⁸ rbc) resulted in better separations than low cell loads (5×10⁷ rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.     

The organization and repair of DNA in the mammalian chromosome. II. A gradient-shape-induced speed dependence affecting DNAs larger than T4 DNA

The addition of a lytic layer to a preformed linear sucrose gradient induces a temporary (up to half a day) initial gradient of considerable steepness which retards the sedimentation of large (>T₄) DNAs. Centrifugation at sufficiently slow angular velocities permits the temporary initial gradient to disappear and therefore the sedimentation distance increases, yielding a rotor speed dependent effect on sedimentation distance. Gradients which are free of this effect are described and shown to permit mouse leukemia cell DNA to sediment independently of rotor speed (5–30 krpm).     

Sedimentation studies of giant DNA molecules present in unsheared whole-cell lysates of D. melanogaster cells.

We have used band sedimentation in shallow density gradients of CsCl in the preparative centrifuge to analyze the distribution of sedimentation coefficients present in tritium labelled DNA from D. melanogaster cells. The cells were lysed according to the method of Kavenoff and Zimm to preserve very high molecular weight DNA. Sedimentation measurements have been carried out as a function of speed of centrifugation. The resulting distribution functions have been interpreted with the aid of the Zimm-Schumaker equation for the speed dependence of the sedimentation coefficient of very high molecular weight DNA. Low-speed centrifugation (3000 rpm) indicates that DNA molecules from the lysate are evenly distributed over values of S_20,w from 0 to 514S. This distribution is very sensitive to changes in speed of centrifugation and is transformed into a bimodal distribution at 12,080 rpm. Analysis of this transformation allows us to postulate that perhaps 55% of the DNA in the lysate may have molecular weights in excess of 40 × 10⁹ g/mol. Some of these molecules may also possess a variety of configurations including partially replicated branched structures.     

Anomalies in the sedimentation behavior of high molecular weight DNA isolated from Proteus mirabilis

The sedimentation properties of a P. mirabilis DNA sample have been investigated at different concentrations and rotor speeds. Pronounced speed effects occurred at high angular velocities. The s value evaluated from low-speed experiments amounts to 61 S., indicating a mean molecular weight of 105 million. Anomalous concentration distributions in the ultracentrifuge cell have been observed at low speeds. At the boundary, a pile-up of concentration occurs which exceeds the total plateau concentration. The concentration elevation decreases with increasing time due to convection which is caused by the existence of a negative density gradient. Despite this convection, accurate mean sedimentation coefficients could be obtained even at extremely low concentrations. A careful analysis of sedimentation coefficient distributions shows, however, that the lending and trailing tails of the boundaries are disturbed by convection. Thus it may be expected that the effect produces difficulties in determining true sedimentation coefficient distributions of polydisperse DNA samples of very high molecular weight.     

DNA relatedness among bacteriophages of the morphological group C3

Six bacteriophages with an elongated head and a short, noncontractile tail were compared by DNA-DNA hybridization, seroneutralization kinetics, mol% G+C and molecular weight of DNA, and host range. Three phage species could be identified. Phage species 1 containedEnterobacter sakazakii phage C2,Erwinia herbicola phages E3 and E16P, andSalmonella newport phage 7–11. These phages had a rather wide host range (4 to 13 bacterial species). DNA relatedness among species 1 phages was above 75% relative binding ratio (S1 nuclease method, 60°C) when labeled DNA from phage C2 was used, and above 41% when labeled DNA from phage E3 was used. Molecular weight of DNA was about 58×10⁶ (C2) to 67 ×10⁶ (E3). The mol% G+C of DNA was 43–45. Anti-C2 serum that neutralizes all phages of species 1 does not neutralize phages of the other two species. Species 2 contains only coliphage Esc-7-11, whose host range was only oneEscherichia coli strain out of 188 strains of Enterobacteriaceae studied; it was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage Esc-7-11 had a base composition of 43 mol% G+C and a molecular weight of about 45×10⁶. Species 3 contains onlyProteus mirabilis phage 13/3a. Its host range was limited to swarmingProteus species. Species 3 was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage 13/3a had a base composition of 35 mol% G+C and molecular weight of about 53×10⁶. It is proposed that phage species be defined as phage nucleic acid hybridization groups.     

Phenotypic differences among the alleles of the T4 recombination defective mutants

Summary The allelic forms of the phage genes T4x and fdsA, as well as T4y and fdsB are compared in terms of their thymidine incorporation in high or low concentrations of thymidine, sensitivity of DNA synthetic capacity to mitomycin C, and sedimentation rates of DNA replicative intermediates. The results show differences among these mutants for the incorporation of thymidine; however all exhibit mitomycin C-sensitive DNA synthesis and have identical aberrant sedimentation rates for their DNA replicative intermediates.     

The nature of the transition mismatches with Watson–Crick architecture: the G*·T or G·T* DNA base mispair or both? A QM/QTAIM perspective for the biological problem

This study provides the first accurate investigation of the tautomerization of the biologically important guanine*·thymine (G*·T) DNA base mispair with Watson–Crick geometry, involving the enol mutagenic tautomer of the G and the keto tautomer of the T, into the G·T* mispair (?G?=?.99?kcal?mol^?1, population?=?15.8% obtained at the MP2 level of quantum-mechanical theory in the continuum with ε?=?4), formed by the keto tautomer of the G and the enol mutagenic tautomer of the T base, using DFT and MP2 methods in vacuum and in the weakly polar medium (ε?=?4), characteristic for the hydrophobic interfaces of specific protein–nucleic acid interactions. We were first able to show that the G*·T?G·T* tautomerization occurs through the asynchronous concerted double proton transfer along two antiparallel O6H···O4 and N1···HN3 H-bonds and is assisted by the third N2H···O2 H-bond, that exists along the entire reaction pathway. The obtained results indicate that the G·T* base mispair is stable from the thermodynamic point of view complex, while it is dynamically unstable structure in vacuum and dynamically stable structure in the continuum with ε?=?4 with lifetime of 6.4·10^?12?s, that, on the one side, makes it possible to develop all six low-frequency intermolecular vibrations, but, on the other side, it is by three orders less than the time (several ns) required for the replication machinery to forcibly dissociate a base pair into the monomers during DNA replication. One of the more significant findings to emerge from this study is that the short-lived G·T* base mispair, which electronic interaction energy between the bases (?23.76?kcal?mol^?1) exceeds the analogical value for the G·C Watson–Crick nucleobase pair (?20.38?kcal?mol^?1), “escapes from the hands” of the DNA replication machinery by fast transforming into the G*·T mismatch playing an indirect role of its supplier during the DNA replication. So, exactly the G*·T mismatch was established to play the crucial role in the spontaneous point mutagenesis.     

Sedimentation of DNA Released from Chinese Hamster Cells

Under high pH and high salt conditions, Chinese hamster cells lyse and release a DNA-containing material of large molecular weight. With increasing lysis time, a smaller material is resolved from the large one. Relative to T4 DNA, the smaller is estimated to be ~2 × 10⁸ daltons (number average). From a comparison of radiation data, the target size of the larger is about 15 times that of the smaller (probably a lower limit estimate). In addition to concentration of alkali, temperature, and time of lysis, the resolution of the smaller from the larger material is shown to be affected by other factors. Three of these are: fluorescent light exposure during lysis, X-irradiation before lysis, and incorporation of actinomycin D before lysis. All of these treatments result in degradation of the smaller molecules if large enough exposures are used. The sedimentation patterns of both DNA materials are strongly speed dependent. This probably results from changes in molecular conformation and concomitant increases in viscous drag with speed. The speed dependence differs qualitatively for the two materials, an observation which suggests that they differ in ways in addition to size.     

Characteristic effect of an anticancer dinuclear platinum(II) complex on the higher-order structure of DNA

It is known that a 1,2,3-triazolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH₃)₂}₂(μ-OH)(μ-1,2,3-ta-N ¹,N ²)](NO₃)₂ (AMTA), shows high in vitro cytotoxicity against several human tumor cell lines and circumvents cross-resistance to cisplatin. In the present study, we examined a dose- and time-dependent effect of AMTA on the higher-order structure of a large DNA, T4 phage DNA (166 kbp), by adapting single-molecule observation with fluorescence microscopy. It was found that AMTA induces the shrinking of DNA into a compact state with a much higher potency than cisplatin. From a quantitative analysis of the Brownian motion of individual DNA molecules in solution, it became clear that the density of a DNA segment in the compact state is about 2,000 times greater than that in the absence of AMTA. Circular dichroism spectra suggested that AMTA causes a transition from the B to the C form in the secondary structure of DNA, which is characterized by fast and slow processes. Electrophoretic measurements indicated that the binding of AMTA to supercoiled DNA induces unwinding of the double helix. Our results indicate that AMTA acts on DNA through both electrostatic interaction and coordination binding; the former causes a fast change in the secondary structure from the B to the C form, whereas the latter promotes shrinking in the higher-order structure as a relatively slow kinetic process. The shrinking effect of AMTA on DNA is attributable to the possible increase in the number of bridges along a DNA molecule. It is concluded that AMTA interacts with DNA in a manner markedly different from that of cisplatin.     

Theory and practice of centrifugal elutriation (CE). Factors influencing the separation of human blood cells

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that selective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5 X 10(8) rbc) resulted in better separations than low cell loads (5 X 10(7) rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.     

Synchronization of mammalian cells by selection and additional chemical block studied by DNA distribution analysis and BrdUrd-Hoechst 33258-technique

Abstract. Ehrlich ascites tumour cells growing in vitro in suspension culture were separated according to volume by the technique of velocity sedimentation in a zonal rotor with a reorienting gradient. Using DNA distribution analysis the sedimentation pattern of the cells could be analysed in detail. With appropriate conditions it was possible to separate pure G1 cells. Samples could also be obtained which were enriched in S or G2 + M cells. The main limitation of the selection in this type of rotor was the reorientation of the gradient which caused disturbances during deceleration of the rotor. The synchronous growth of selected G1 cells has been studied in detail to investigate the reasons for the rather poor synchrony of these cells. The poor synchrony was found to be caused mainly by the small volume of the selected G1 cells compared with the normal volume of G1 cells in an asynchronous population. The synchronization of these cells could be essentially improved by a short treatment with excess thymidine causing a metabolic block at the G1/S border. The duration of this treatment could be minimized using DNA distribution analysis of growing cells after releasing of the block. The durations of the cell cycle phases in synchronized cells agreed with the values calculated in asynchronous cells by DNA distribution analysis and the BrdUrd-Hoechst 33258-technique.     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta™ Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas.To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta™ Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report [Cancer Res, 49 (1989) 5305]. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively.These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.