A critical examination of possible fractionations of human DNA according to base composition.

Human DNA has been fractionated according to base composition by sedimentation equilibrium in an HgCl2/Cs2SO4 density gradient, followed by sedimentation equilibrium in an actinomycin/cesium formate density gradient. The fractions of different base composition resulting from this procedure were subsequently analyzed by sedimentation equilibrium in CsCl, DNA renaturation kinetics, and electron microscopy. All fractions contain similar kinetic classes of repeated DNA sequences as judged by renaturation studies. Short (300 nucleotides) interspersed repeated sequences are found in all fractions with no noticeable enrichment for these sequences in any fraction. Repeated sequences from fractions of different base composition are partially able to cross-hybridize, demonstrating that nearly identical repeated sequences occur in molecules of different base composition. These findings are critically compared to reports of successful density gradient fractionations of different human DNA sequence classes.     

Cyclosporin synthetase is a 1.4 MDa multienzyme polypeptide. Re-evaluation of the molecular mass of various peptide synthetases.

The earlier determined molecular mass of 0.8 MDa for the multifunctional polypeptide, cyclosporin synthetase, was re-evaluated by SDS-PAGE and CsCl density gradient centrifugation. In SDS-PAGE, new molecular mass values as standards were available from sequencing data. In the CsCl density gradient extremely low protein concentrations, such as 10-50 nM could be analysed due to the fluorescence detection system of the analytical ultracentrifuge. Both methods yielded approximately the same value of about 1.4 MDa. Using this molecular mass of cyclosporin synthetase as a reference the molecular masses of various related enzymes could be re-evaluated in SDS-PAGE. The sedimentation coefficient of 26.3 S for cyclosporin synthetase indicates an oblate overall shape of the enzyme.     

Further characterization of intracytoplasmic A particle-associated DNA.

A DNA species with buoyant densities greater than mouse cellular DNA was found associated with intracytoplasmic A particles (CAP) isolated from mouse mammary tumor virus-infected mouse mammary tumors and mouse Leydig cell tumors which produce CAP but no complete mouse mammary tumor virus virions. This DNA species was absent in identically prepared tissue fractions from tumors which did not contain CAP. Treatment of CAP-associated DNA with pancreatic RNase A did not alter the buoyant density although a reduction in apparent molecular weight (broadening of the DNA band at equilibrium) was observed upon analytical equilibrium sedimentation in CsCl. The molecular weight of untreated CAP-associated DNA was estimated to range from 0.8 x 10(6) to 3.1 x 10(6). Base composition analysis showed CAP-DNA to possess an approximate guanine plus cytosine content of 38%. Ninety percent of CAP-associated DNA eluted as single-stranded molecules upon hydroxyapatite column chromatography, a characteristic that accounts in part for its higher buoyant density in neutral CsCl compared to native double-stranded mouse DNA. In two preparations, CAP-DNA had a sedimentation coefficient of 7 to 8S.     

Isolation of plasmid deoxyribonucleic acid from two strains of Bacteroides.

Two clinical isolates of Bacteroides contained covalently closed circular deoxyribonucleic acid (DNA) as shown by sedimentation in an alkaline sucrose gradient, CsCl ethidium bromide equilibrium centrifugation, and electron microscopy. Bacteriodes fragilis N1175 contained a homogeneous species of plasmid DNA with a molecular weight of 25 x 10(6). Bacteroides ochraceus 2228 contained two distinct, covalently closed circular DNA elements. The larger cosedimented with the covalently closed circular DNA form of the R plasmid, R100, corresponding to a molecular weight of 70 x 10(6); the smaller sedimented as a 58S molecule with a calculated molecular weight of 25 x 10(6). The roles of these plasmids are unknown. Neither strain transferred antibiotic resistance to plasmid-negative Bacteroides or Escherichia coli, and neither produced bacteriocins active against other Bacteroides or sensitive indicator strains of E. coli.     

Replication Process of the Parvovirus H-1 II. Isolation and Characterization of H-1 Replicative Form DNA

The Parvovirus H-1 replicates autonomously in hamster embryo cells. A DNA synthetic event, called HA-DNA synthesis, upon which subsequent viral RNA and viral hemagglutinin synthesis is dependent, is initiated in late S phase of the infected cell (18). It was postulated that HA-DNA represents parental viral replicative form DNA (RF DNA). This study describes the isolation and characterization of H-1 RF DNA as part of the continuing study of the mechanisms and control of DNA replication in the eukaryotic cell. The H-1 RF DNA is a linear duplex molecule containing the viral strand and its complement. The complementary strands of the RF DNA have been separated by equilibrium density gradient centrifugation. The RF DNA has a buoyant density of 1.705 in neutral CsCl and an estimated guanine plus cytosine (GC) content of 45.9%. It has a sedimentation coefficient of 17S. The calculated molecular weight of 3.7 x 10(6) is twice that of the single-stranded virion DNA. H-1 virions contain DNA that is homogeneous and free of complementary strands.     

Structure and synthesis of a lipid-containing bacteriophage. The molecular weight and other physical properties of bacterophage PM2.

Several physical and chemical parameters of bacteriophage PM2 have been measured. The sedimentation constant was determined to be s-20,w=293 S. The buoyant density in sucrose at 20 degrees C was 1.24 g cm+-3 and in CsCl at 25 degrees C was 1.29 g cm-3. The high-speed equilibrium centrifugation method of Yphantis (1964) was used to measure the molecular weight of PM2. The necessary auxiliary parameters were also determined. A value of 0.771 plus or minus 0.005 cm-3 g-1 for the apparent specific volume at constant chemical potential in 1 M sodiium chloride has been obtained by pycnometry; the viral concentration was determined using the absorption coefficient at 260 nm (4.60 plus or minus 0.10 cm-2 mg-1), which in turn was calculated from the phosphorous content of the virus (17.89 plus or minus 0.28 mu-g of P per mg dry weight dry weight of virus). The molecular weight of PM2 determined with these parameters is (44.1 plus or minus 1.2 x 10-6). From the phosphorous content of the virus, the percentage of phosphorous known to be in its DNA (Camerini-Otero and Franklin, 1972), and the molecular weight of the bacteriophage, we have calculated a molecular weight for PM2 DNA of 6.26 x 10-6, which confirms values determined using empirical relationships.     

Analysis by isopycnic centrifugation of isolated nucleoids of Escherichia coli.

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 +/- 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 +/- 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.     

Characteristics of a new bacteriophage, Psp231a, infecting Pseudomonas phaseolicola HB10Y.

Bacteriophage Psp231a infects Pseudomonas phaseolicola, strain HB10Y, which is the host cell for the enveloped bacteriophage phi 6. This paper describes the biophysical characteristics of Psp231a and the physical properties of its nucleic acid. In electron micrographs the virion appears as an icosahedral structure, approximately 55 nm in diameter, with a short tail. The virion density is 1.48 g/cm3 in CsCl, and the sedimentation coefficient is approximately 407S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 12 polypeptides ranging in molecular weight from 5,000 to 117,000. The nucleic acid of Psp231a is linear, double-stranded DNA of molecular weight 28 X 10(6). Its density in CsCl is 1.716 g/cm3, and its sedimentation coefficient in 3 M CsCl is 20.0S, corresponding to an S020,W of 34S.     

Characteristics of DNA in vegetative specimens of Bursaria truncatella]

DNA from Bursaria truncatella was isolated and purified by conventional methods. The DNA base content was calculated from both centrifugation in CsCl and melting. The GC-content is 24%. In CsCl density gradient 3H-DNA is banded as a single peak at a range 1.682--1.688 g/cm3 with the maximum at 1.684 g/cm3. The Tm in 0.12 M FB (pH 6.8) was 79 degrees C. About 50% of DNA seems to be represented by highly repetitive sequences, another 50% being made of single-copy sequences (a preliminary data on DNA-DNA reassotiation kinetics). For the estimation of the molecular weight of DNA, the cells were lysed immediately before the centrifugation, at the surface of the alkaline isokinetic sucrose gradient solution. Two components of DNA were detected with molecular weights 10-10(6) and 100-10(6) daltons. It is likely that these two components belong to the macronuclear DNA, because according to cytophotometrical evidence the DNA content in the macronucleus of B. truncatella is 2500 times as much as that in its micronucleus.     

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.     

Sedimentation equilibrium of DNA samples heterogeneous in density

The problem of determining the molecular weight of DNA samples by sedimentation equilibrium in a buoyant-density gradient is considered for the case of DNA samples with density heterogeneity. By determining apparent molecular weights in two or more buoyant mediums, quantitative measure of the amount of density heterogeneity can be determined. This method may be employed to determine both the true molecular weight and the extent of base composition heterogeneity.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: II. Detection and Characterization of Simian Virus 40 Pseudovirions

Purified simian virus 40 (SV40) virions, grown in primary African green monkey kidney cells labeled prior to infection with (3)H-thymidine, contain a variable quantity of (3)H-labeled deoxyribonucleic acid (DNA). This DNA is resistant to deoxyribonuclease, sediments at 250S, and is enclosed in a particle that can be precipitated with SV40-specific antiserum. DNA-DNA hybridization experiments demonstrate that this (3)H-labeled component in purified SV40 virions is cellular DNA. When this (3)H-labeled DNA is released from purified virus with sodium dodecyl sulfate, it has an average sedimentation constant of 14S. Sedimentation through neutral and alkaline sucrose gradients shows that this 14S DNA is composed of a collection of different sizes of DNA molecules that sediment between 11 and 15S. As a result of this size heterogeneity, SV40 virions containing cellular DNA (pseudovirions) have a variable DNA to capsid protein ratio and exhibit a spectrum of buoyant densities in a CsCl equilibrium gradient. Pseudovirions are enriched, relative to true virions, on the lighter density side of infectious SV40 virus banded to equilibrium in a CsCl gradient. Little or no cellular DNA was found in purified SV40 virus preparations grown in BSC-1 or CV-1 cells.     

Analysis of Euglena gracilis chloroplast deoxyribonucleic acid with a restriction endonuclease, EcoRI.

Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoRI) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight of the circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosomal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.     

Concentration dependence in three-component systems in sedimentation equilibrium in buoyant density gradients

The data on the band widths and band shapes of several DNA's at various concentrations in sedimentation equilibrium in a CsCl density gradient have recently become available. In the present report, these literature data are treated in the following manner: (1) based on a theory of isotope-substitution, calculations are made of the molecular weights at infinite dilution, and (2) to explain the concentration dependence of band widths and band shapes, a theory of charge and hydration is put forth, and it is shown that by retaining the terms involving the charge of the macromolecules, it is possible to account for most of the concentration dependence.     

Fractionation of a hyaluronic acid preparation in a density gradient. Some properties of the hyaluronic acid

1. Hyaluronic acid was isolated from ox synovial fluid by sedimentation equilibrium in a caesium chloride density gradient (Silpananta, Dunstone & Ogston, 1967). The product was almost free from chondroitin sulphate and from protein. 2. Its composition did not differ significantly from that of the carbohydrate part of the protein-containing material isolated by filtration. Its physicochemical properties and molecular configuration were similar, except for its viscosity, which showed markedly reduced concentration-dependence and shear-dependence. This suggests that the associated protein tends to form links between molecules of hyaluronic acid. 3. The accurate measurement of viscosity at very low velocity gradient, by use of the damping of oscillations in a Couette viscometer, is described. 4. A method is described for measuring, approximately, the thermodynamic non-ideality of a solute from the shape of its schlieren curve at sedimentation equilibrium in a density gradient. 5. A value for the partial specific volume of hyaluronic acid in dilute salt solution was calculated from its isopycnic density in a caesium chloride gradient.     

Isolation of viral RNA using cesium chloride-ethidium bromide equilibrium sedimentation

The effect of ethidium bromide (EB) on the buoyant density of reovirus RNA during equilibrium sedimentation has been investigated. The addition of the dye ethidium bromide was found to reduce the buoyant density of reovirus RNA in a Cs₂SO₄ gradient by a value of 0.13 to 0.15 g/cc, and provided a separation limit of 0.10 g/cc relative to the ? of marker DNA. Ethidium bromide was found also to reduce the ? of reovirus RNA to allow this RNA to band on a CsCl gradient. The separation factor between DNA and RNA on a CsCl-EB gradient was found to be 0.23 g/cc, indicating this type of gradient to be highly effective for separating the two types of polynucleotides.     

The proteoglycans of bovine nasal cartilage and human articular cartilage: a sedimentation equilibrium analysis

Cartilage proteoglycan was isolated from bovine nasal septum and fractionated according to buoyant density after dissociative CsCl density gradient centrifugation. Gel-exclusion chromatography showed that hyaluronic acid was present in fractions of density lower than 1.69 g/mL. The molecular weight, assessed by sedimentation equilibrium analysis, of the proteoglycan present in the fractions with density > 1.69 g/mL, which appeared chromatographically homogeneous and constituted 54% of the preparation, ranged from 1.0 to 2.6 × 10⁶ for v = 0.55 cm³ g^?1. Carbodiimide-induced modification of the carboxyl groups by methylamine resulted in a reduction of the molecular weight to 0.74 – 1.25 × 10⁶. An analogous reduction in molecular weight was obtained after equilibration of this proteoglycan fraction with hyaluronic acid oligomers containing five disaccharide units. Since both procedures are known to cause inhibition of the interaction between proteoglycans and hyaluronic acid, it is suggested that this lower molecular-weight range represents the true degree of polydispersity of the sub-units of hyaline cartilage proteoglycan constituting this fraction, while the higher values obtained for the intact proteoglycan are the result of the presence of hyaluronic acid in the sample. The molecular-weight range of the whole proteoglycan subunit preparation, assessed after carboxyl group modification, was 0.5–1.2 × 10⁶. Apparently normal and abnormal cartilage was excised from single human osteoarthrosic femoral heads. Proteoglycans extracted by 4M guanidine hydrochloride were isolated after dissociative density gradient centrifugation and subjected to carboxyl group modification. Preparations from normal tissue exhibited molecular-weight averages ranging from 5 to 9 × 10⁵. A molecular-weight reduction was observed with proteoglycans isolated from abnormal areas.     

Mitochondrial DNA of Yoshida ascites tumour cells. Physicochemical properties and biological function.

Mitochondrial DNA from Yoshida A.H. 130 cells, has been characterized by determination of the buoyant density by CsCl equilibrium density gradient centrifugation and the thermal denaturation and renaturation behaviour. These studies have been carried out parallelly on nuclear DNA from the same cells in order to search for possible differences between both DNAs. Mitochondrial DNA of Yoshida cells presents an equilbrium in CsCl of 1.7154 g/cm3 and a sharp melting with a Tm of 92 degrees C. Nuclear DNA presents an equilibrium of 1.7030 g/cm3 and a Tm of 88 degrees C. The guanine plus cytosine content in both DNAs has been calculated from tumour results and compared with the content in normal rat liver cells M-DNA of tumour cells presents a higher guanine plus cytosine content than N-DNA, whereas in normal liver cells is higher in N-DNA. N-DNAs of both normal and tumour cells have the same guanine plus cytosine content, whereas M-DNA from tumour cells presents a significant increase (about 35%) with regard to this from normal liver cells.     

Isolation of a DNA fraction highly enriched in transfer RNA genes from Xenopus laevis.

A DNA fraction highly enriched in tRNA genes can be isolated from the Xenopus laevis genome by the use of Ag+/Cs2SO4 density gradients. Ag+ shows a low affinity for some tRNA cistrons, allowing their separation from bulk DNA upon equilibrium centrifugation in a Cs2SO4 density gradient. Contaminating DNA in the resulting tDNA fraction is further removed by two additional CsCl density gradient centrifugations. The final DNA fraction is 60-fold enriched in tRNA genes, compared to the starting DNA material.     

Use of preparative CsCl gradients to estimate DNA buoyant densities: potential errors due to fractionation artifacts

The equilibrium banding behavior of high molecular weight DNA has been examined after centrifugation in CsCl gradients on angle rotors in the Spinco preparative ultracentrifuge. DNAs of base composition ranging from 30% GC to 71% GC, from a variety of species, band in close agreement to a calculated curve relating density and radial distance in angle preparative gradients, but only if the DNA is sheared prior to centrifugation. Unsheared DNA, after bottom tube puneture and drop collection by conventional methods, displays a characteristic unsymmetrical profile and the fraction with the largest concentration of DNA appears in a much lower fraction number than expected. This artifact leads to misealculation of buoyant densities from preparative gradients and interferes with fractionation of DNA, as for instance in separating satellite bands. Collection of the gradient from the top reverses the direction of the profile asymmetry and shearing eliminates it entirely.