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.     

Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.     

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.     

Equilibrium sedimentation of a polyelectrolyte in a density gradient of a low-molecular weight electrolyte. I. DNA in CsCl

Previous work had indicated that the molecular weight calculated from the equilibrium distribution of DNA in a CsCl density gradient appears to be one half the estimated value of the true molecular weight. In the present study, the sedimentation equilibrium of a polyclectrolyte in a density gradient generated by a low molecular weight electrolyte is treated in terms of the representation according to which a constant fraction of the counterions is considered permanently immobilized and the remaining fraction completely free. Equations for the preferential hydration and the molecular weight are obtained. The validity of the treatment is tested by applying it to experimental data on the equilibrium sedimentation of ?X 174 DNA in CsCl, and it is found that the molecular weight calculated in this way is in agreement with the accepted value for this molecule. Also, recalculation of published data on density gradient centrifugation of T2 DNA in CsCl according to the present treatment brings up the molecular weight to within the range of values given by other methods.     

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.     

Using analytical ultracentrifugation to study compositional variation in vertebrate genomes

Although much attention has recently been directed to analytical ultracentrifugation (AUC), the revival of interest has hardly addressed the applications of this technology in genome analysis, and the extent to which AUC studies can quickly and effectively complement modern sequence-based analyses of genomes, e.g. by anticipating, extending or checking results that can be obtained by cloning and sequencing. In particular, AUC yields a quick overview of the base compositional structure of a species' genome even if no DNA sequences are available and the species is unlikely to be sequenced in the near future. The link between AUC and DNA sequences dates back to 1959, when a precise linear relation was discovered between the GC (guanine+cytosine) level of DNA fragments and their buoyant density in CsCl as measured at sedimentation equilibrium. A 24-hour AUC run of a high molecular weight sample of a species' total DNA already yields the GC distribution of its genome. AUC methods based on this principle remain sensitive tools in the age of genomics, and can now be fine-tuned by comparing CsCl absorbance profiles with the corresponding sequence histograms. The CsCl profiles of vertebrates allow insight into structural and functional properties that correlate with base composition, and their changes during vertebrate evolution can be monitored by comparing CsCl profiles of different taxa. Such comparisons also allow consistency checks of phylogenetic hypotheses at different taxonomic levels. We here discuss some of the information that can be deduced from CsCl profiles, with emphasis on mammalian DNAs.     

Nucleotide Composition of Nuclear and Mitochondrial Deoxyribonucleic Acid of Fungi

The buoyant density of nuclear and mitochondrial deoxyribonucleic acid (DNA) from 14 species of fungi was determined by CsCl density gradient equilibrium centrifugation. The buoyant density of both types of DNA was the same for all three Mucorales analyzed. The buoyant density of mitochondrial DNA was significantly lower than that of the nuclear DNA for nine species of Ascomycetes and two species of Basidiomycetes. No simple correlation could be obtained from the comparison of the two types of DNA. Mitochondrial DNA represented a very small proportion of total DNA. Heat-denatured mitochondrial DNA renatured more readily than nuclear DNA.     

Specific Labeling and Physical Characterization of R-Factor Deoxyribonucleic Acid in Escherichia coli

The molecular nature of R-factor deoxyribonucleic acid (DNA) was examined in Escherichia coli by using a method for the specific labeling of the derepressed R factor, R1, in a female cell after conjugation. Sixty minutes after mating, the R factor was isolated as a single molecule with a molecular weight of 65 x 10(6) daltons. This single molecular species sedimented as either a covalently closed molecule or a "nicked" circle. When the single R-factor component was centrifuged in a CsCl density gradient, only a single homogeneous species with a buoyant density of 1.711 g/cm(3) was observed. R-factor DNA was also isolated directly from exponentially growing cells of E. coli as a covalently closed single molecular species comprising about 1% of the total cellular DNA. Previous studies in Proteus show that R1 factor DNA components of buoyant density 1.709, 1.711, and 1.716 g/cm(3) can be identified as distinct replicons. It is suggested that the single molecule of R1 observed in E. coli is most simply explained as a composite structure resulting from a recombinational assemblage of a 1.709 and 1.716 g/cm(3) replicon.     

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.     

Long-chain triglyceride lipases of pig liver

The blood-group specific glycoproteins of human ovarian cyst fluids have been isolated by equilibrium density gradient centrifugation in CsCl; they have been characterised in terms of buoyant density, selective salvation and apparent molecular weight, both in CsCl and Cs(2)SO(4).     

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.     

Partial denaturation of mouse DNA in preparative CsCl density gradients at alkaline pH.

A new technique--partial denaturation of DNA in equilibrium CsCl density gradients at pH 11.4--is used to determine the distribution of intermediate states in the melting of mouse DNA. When the technique is applied in the preparative ultracentrifuge, the DNA is fractionated according to stability. Neutralization of the partially denatured fractions results in the recovery of most of the DNA in its native form. The individual fractions are more homogeneous than the total DNA: they have decreased density heterogeneity (smaller band widths), neutral CsCl buoyant densities that differ from the average, and more homogeneous melting profiles with melting temperatures that differ from the average.     

Isolation and Characterization of Kinetoplast DNA Networks and Minicircles from Crithidia fasciculata*

SYNOPSIS. Covalently closed kinetoplast DNA networks have been isolated from stationary phase Crithidia fasciculata cells by a technic involving selective pelleting of the networks at a low centrifugal field. Approximately 62% of the kinetoplast DNA of the cell was recovered free of nuclear DNA by simple differential centrifugation. Purified kinetoplast DNA networks were visualized both in the electron microscope and in the light microscope. Closed networks sedimented as a homogeneous band both in neutral and alkaline sucrose, with an s_20w in neutral sucrose of approximately 5 × 10³. Closed monomeric minicircles were isolated from purified networks by mild sonication and band sedimentation in alkaline sucrose. Several physical properties of closed monomeric minicircles were measured. These included molecular weight, buoyant density in CsCl, superhelix density and sedimentation coefficient.     

Density gradient analysis of newly replicated DNA from synchronized mouse lymphoma cells

The replication of DNA in synchronous cultures of mouse lymphoma cells was investigated by use of CsCl density gradient centrifugation. We found that the buoyant density of newly replicated DNA depended upon the particular stage of S phase in which synthesis occurred. In early S phase, newly replicated DNA exhibited buoyant densities which were slightly higher, on the average, than that of pre-existing DNA. As S phase progressed, newly replicated DNA shifted to lower buoyant densities, until, near the end of S phase, densities less than pre-existing DNA were observed. These observations are discussed in terms of their possible relevance to base compositional differences between nucleotide sequences made in early as opposed to middle or late S phase.     

Purification and Biophysical Properties of Acute Haemorrhagic Conjunctivitis Virus

The buoyant density of acute haemorrhagic conjunctivitis virions labeled with either [(3)H]uridine or [(3)H]leucine was 1.34 g/ml in CsCl and 1.25 g/ml in sucrose. RNA extracted from the virions gave a sedimentation coefficient of approximately 34S in sucrose, and was found to be sensitive to RNase. Molecular weight of RNA was calculated to be 2.5 x 10(6) using poliovirus RNA for reference.     

The gene transfer agent of Rhodopseudomonas capsulata,: Purification and characterization of its nucleic acid

Photosynthetic bacteria of the species Rhodopseudomonas capsulata are capable of exchanging genetic information via a recently discovered gene transfer agent (GTA). The 70S particle mediating the genetic exchange was purified and its nucleic acid was analyzed. Cell-free filtrates containing GTA were prepared by filtration of stationary cultures of R. capsulata on an Amicon thin-channel filtration apparatus. Purification of this filtrate was achieved by successive membrane filtration, diafiltration, agarose-gel filtration, ion-exchange chromatography on diethylaminoethyl cellulose, and sucrose gradient sedimentation, resulting in an overall purification of 4000-fold with a yield of 2–4%. [³H]thymidine-labeled nucleic acid isolated from this material was identified as deoxyribonucleic acid on the basis of its resistance to alkaline hydrolysis and its buoyant density of 1.718 g/ml in CsCl. The double-stranded nature of the deoxyribonucleic was demonstrated by its resistance to degradation by the single-strand-specific S₁-nuclease and the density shift in CsCl of +0.016 g/ml upon denaturation. Its molecular weight was estimated to be 3.6 × 10⁶ from sucrose gradient sedimentation in the presence of markers, and the linear, unnicked nature of the molecule was evident from sedimentation in an alkaline sucrose gradient.     

In Vivo Conversion of the Single-Stranded DNA of the Kilham Rat Virus to a Double-Stranded Form

Kilham rat virus (KRV) contains linear, single-stranded DNA in the virion. The fate of radioactive viral DNA was followed after infection of monolayer cells. Within 60 min after infection of cells, 28 to 42% of the parental viral DNA is converted to a new form. This new DNA form is believed to be double stranded and linear on the basis of its sedimentation in neutral and alkaline sucrose gradients, elution from hydroxyapatite columns, its buoyant density in equilibrium CsCl density gradients, and appearance in the electron microscope. The double-stranded linear KRV DNA may be analogous to the replicative form of certain bacteriophages, including phiX174, which contain single-stranded circular genomes.     

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.     

Isolation and characterization of Caulobacter crecentus bacteriophage phi Cd1.

A DNA-containing bacteriophage, phiCd1, was isolated from sewage and shown to infect both stalked and swarmer cells of Caulobacter crescentus strain CB13B1a. phiCd1 is a small, icosohedral bacteriophage, 60 nm in diameter, which possesses a short, noncontractile tail, 10 to 12 nm in length. The bacteriophage particle is composed of at least eight structural proteins. phiCd1 nucleic acid exists as a linear duplex of DNA as judged by: (i) thermal denaturation (Tm), (ii) CsCl density gradient centrifugation, and (iii) chemical analysis of its base composition. The DNA is 61% guanosine plus cytosine, has a buoyant density in CsCl of 1.721 +/- 0.001 g/cm3, and denatures sharply at 78.5 C in 0.1 SSC (standard saline citrate) buffer. The S20, w value for the DNA is 34.3 +/- 0.1S as compared with T7 DNA, indicating a molecular weight of about 29 x 10(6).     

Properties of Thermoactinomyces vulgaris phage Ta1 and its extracted DNA

The virulent phage Ta1 was obtained in good yields from infected cultures of Thermoactinomyces vulgaris 1227. The purified phage was found to sediment with a single band, the sedimentation constant being (519 +/- 14)S, and to exhibit a typical nucleoprotein behaviour in UV-spectrophotometric and CD experiments. The Ta1 phage consists of a hexagonal head about 0.056 micrometers in diameter and a very short tail. It is morphologically similar to the temperate Salmonella phage P22. The nucleic acid extracted from the phage was found to be a double-stranded linear DNA with a G+C content of 42 mole-% as deduced both from its melting temperature and buoyant density in CsCl. Analytical sedimentation revealed a high degree of molecular homogeneity of Ta1 Dna. the sedimentation constant of this DNA amounts to (35.9 +/- 0.3)S, corresponding to a DNA molecular weight of about 29 millions daltons. The biological activity of Ta1 DNA was indicated by its ability to infect the mycelium of the components T. vulgaris strain 1227 and to give rise to mature phages.