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.     

Characterization of the Kilham Rat Virus

Kilham rat virus (KRV) was found to grow in a rat nephroma cell line and to form plaques on secondary rat embryo monolayers. The virus was purified by enzymatic treatment and isopycnic cesium chloride sedimentation. KRV bands at a density of 1.41 g/cm(3) in cesium chloride. It contains about 26.5% deoxyribonucleic acid (DNA). The sedimentation coefficient S(20,w) in sucrose gradients was 122 corresponding to a molecular weight of 6.6 x 10(6) daltons. The reaction of formaldehyde with the KRV virion suggests that the DNA in situ is single-stranded. DNA extracted from KRV had a buoyant density of 1.715 g/cm(3) in cesium chloride. The S(20,w) was determined in sucrose gradients to be 16, and the molecular weight was calculated to be approximately 1.7 x 10(6) daltons. The base composition of the DNA is 26.7% adenine, 30.8% thymine, 20.0% guanine, and 22.5% cytosine. On the basis of its noncomplementary nucleotide ratio, melting curve, and the reaction with formaldehyde, the DNA of KRV is believed to be single-stranded.     

DNA contained by two densonucleosis viruses.

The DNA contained by particles of densonucleosis viruses 1 and 2 were analyzed within the particle, and properties of DNA extracted from these particles were determined. The DNA appears to exist as a single-stranded molecule with limited secondary structure within particles, as assessed by spectral changes induced by formaldehyde, melting profiles, and circular dichroism studies. The single-stranded DNA had an apparent molecular weight of 1.9 X 10(6) to 2.2 X 10(6) as assessed by differences in the molecular weight of virus particles and top component and percentage of nucleic acid. DNA extracted from virus particles in low-salt buffers possessed properties typical of a single-stranded molecule. Double-stranded DNA could be extracted from virus particles under appropriate high salt and elevated temperature. The linear double-stranded DNA extracted from both viruses had a molecular weight of about 3.9 X 10(6) to 4.1 ZX 10(6) determined by neutral sedimentation and electron microscopy and an equivalent genome size determined by reassociation kinetics. About 87% of the DNA was homologous between the two viruses.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.     

Infection of peripancreatic lymph nodes but not islets precedes Kilham rat virus-induced diabetes in BB/Wor rats.

A parvovirus serologically identified as Kilham rat virus (KRV) reproducibly induces acute type I diabetes in diabetes-resistant BB/Wor rats. The tissue tropism of KRV was investigated by in situ hybridization with a digoxigenin-labelled plasmid DNA probe containing approximately 1.6 kb of the genome of the UMass isolate of KRV. Partial sequencing of the KRV probe revealed high levels of homology to the sequence of minute virus of mice (89%) and to the sequence of H1 (99%), a parvovirus capable of infecting rats and humans. Of the 444 bases sequenced, 440 were shared by H1. KRV mRNA and DNA were readily detected in lymphoid tissues 5 days postinfection but were seldom seen in the pancreas. High levels of viral nucleic acids were observed in the thymus, spleen, and peripancreatic and cervical lymph nodes. The low levels of infection observed in the pancreas involved essentially only endothelial and interstitial cells. Beta cells of the pancreas were not infected with KRV. These findings suggest that widespread infection of peripancreatic and other lymphoid tissues but not pancreatic beta cells by KRV triggers autoimmune diabetes by perturbing the immune system of genetically predisposed BB/Wor rats.     

Properties of the histidines of chymotrypsinogen: comparison with alpha-chymotrypsin

Adeno-associated virus linear, single polynucleotide chains contain an inverted terminal repetition which allows the formation of single-stranded circles when the DNA is exposed to annealing conditions. Under appropriate annealing conditions single-stranded circular dimers are formed, the majority of which have two projections separated by 180 ° visible in the electron microscope. We conclude that these projections represent the regions of self-complementarity (inverted terminal repetition) contained within the virus DNA. Measurements of the lengths of the projections indicate that the length of the inverted terminal repetition represents approximately 1.5% of the genome.     

Secondary infection of rat lungs with Pasteurella pneumotropica after Kilham rat virus infection

Lung congestion was observed after an outbreak of Kilham rat virus infection (KRV) in a rat colony, previously free of all rat viruses. A high proportion of congested lungs contained Pasteurella pneumotropica suggesting that KRV might have caused primary damage to the alveoli (hitherto not recorded) which allowed the secondary bacterial colonization. Experimental infection of rats with KRV caused acute damage to the lung alveoli. Since KRV infection is very common in animal facilities it could therefore be a significant agent in the development of respiratory disease.     

DNA of a new parvo-like virus isolated from the silkworm,Bombyx mori

Parvo-like virus, which was designated as “Ina-flacherie virus (Ina-FV),” was isolated from the silkworm, Bombyx mori, and the properties of its DNA were characterized. Purified Ina-FV had a diameter of 22 ± 0.5 nm and a sedimentation coefficient of 102 S. On density gradient separation in CsCl, particles were found at densities of 1.40 and 1.45 g/ml. The DNA content of Ina-FV was 28 ± 2%. The DNA in low-salt buffer possessed properties typical of a single-stranded (ss) molecule. Double-stranded (ds) DNA was extracted under conditions of appropriate high salt and elevated temperature. Electron microscopical examination revealed that the ds DNA was composed of linear molecules with an average length of 1.7 μm and other less well-defined structures. The linear ds molecule had a molecular weight of about 3.4 × 10⁶ determined by electron microscopy (EM) and agarose gel electrophoresis. When the ds DNA was alkali-denatured and examined in an EM, linear ss molecules with approximate length of 1.7 μm were observed, indicating that the linear ds molecule was formed from the annealing of the linear ss molecules of unit length. These data suggest that Ina-FV is closely related to members of the densovirus subgroup.     

Characterization of the protein and nucleic acid of Aleutian disease virus.

Aleutian disease virus (ADV) was extracted and purified from infected mink. Nucleic acid extracted from the virus was examined in an electron microscope. Three different sizes of molecule, with approximate lengths of 1.2, 0.55, and 0.25 micron, were observed. The ratios of the large molecules to the small molecules were similar in all the particles prepared under different conditions. Equilibrium CsCl density gradient centrifugation showed that ADV nucleic acid had a buoyant density of 1.733 g/cm3. In Cs2SO4, ADV had a lower buoyant density than that of double-stranded RNA. These properties and its sensitivity to DNase suggested that ADV contains DNA. Thermal denaturation curves revealed that the DNA of ADV had a single-stranded configuration. Polypeptide analysis of ADV by polyacrylamide gel electrophoresis revealed the presence of four polypepties, with molecular weights of 30,000, 27,000, 20,500, and 14,000. These polypeptides were present in a ratio of 10:3:10:1, respectively. The data suggested that ADV is closely related to the members of the parvovirus groups.     

DNA Polymerase Activity associated with Purified Kilham Rat Virus

RNA tumour viruses contain an enzyme which can transcribe DNA from an RNA template^1,2, an endonuclease and a DNA-dependent DNA polymerase activity^3,4. RNA polymerase has been reported in vaccinia virus^5,6, reovirus^7,8 and cytoplasmic polyhidrosis virus⁹. I wish to describe a DNA polymerase activity associated with a highly purified preparation of the parvovirus, Kilham rat virus (KRV), which is thus the first report of a DNA polymerase associated with a DNA virus. KRV, a small virus first isolated from a rat sarcoma¹⁰, is antigenically related to the H viruses isolated from human transplantable tumours¹¹. Those parvoviruses which have been characterized all contain single stranded DNA with molecular weights of 1.5 to 2.5 × 10⁶ (refs. 12,13 and 14).     

Electron microscopic analysis of replicating DNA of sea urchin embryos

DNA was extracted from Paracentrotus lividus embryos at the third S phase after fertilization and analyzed with the electron microscope. The most relevant structures observed in this actively replicating DNA are clusters of short, closely spaced microbubbles (about 0.1 μm long on the average), partially or entirely single-stranded molecules and few linear forks. Unexpectedly, no long eye forms were observed. The analysis of DNA purified from gastrulae and from adult somatic tissues has revealed the same structures, although at a low frequency. A quantitative analysis has been carried out to determine the size distribution and spacing of microbubbles. A number of control experiments have been performed to characterize these structures better. Various possibilities are discussed to account for the presence of the observed forms and the absence of larger eyes.     

A nearby inverted repeat of the terminal sequence of herpes simplex virus DNA.

When herpes simplex virus DNA is digested with λ-exonuclease, annealed, and then mounted for observation in the electron microscope, two types of molecules are seen. One type is circular DNA which forms because the enzyme has revealed the terminal repetition. The other type is full length linear duplex DNA with a short single-stranded loop on one end. This latter type, which apparently represents a fold-back reaction, forms because there is a nearby inverted repeat of the terminal sequence of herpes simplex virus DNA.     

Electron microscopy of the segmented RNA genome ofLa Crosse virus: absence of circular molecules.

The three species of single-stranded RNA present in La Crosse virus were examined in the electron microscope. Because large amounts of contaminating cellular DNA are copurified with the virus despite extensive attempts to purify the virus, it was necessary to use procedures that eliminated the bulk of this DNA before the viral RNA was analyzed. When this was done, the modal lengths of La Crosse virus RNA were 0.4, 2.0, and 3.1 mum. These lengths correspond well to their known molecular weights of 0.4 x 106, 1.8 x 106, and 2.9 x 106. Under the denaturing conditions used to permit complete spreading of these single-stranded RNA molecules, no single-stranded circular molecules are observed. Therefore, the circular nucleocapsids present in La Crosse virus and some other bunyaviruses do not appear to be due to convalent linkage of the ends of the RNA genome.     

Double-Length, Circular, Single-Stranded DNA from Filamentous Phage

Wild-type and gene 3 mutant filamentous phage stocks, containing different relative amounts of multiple-length particles, were treated exhaustively with DNase and then were highly purified. The phage DNA was extracted and examined by electron microscopy. In all cases, about 0.03% of the molecules were circular dimers. (3)H-labeled phage DNA was separated as to size by sedimentation in a preformed CsCl density gradient. Individual fractions were then examined in the electron microscope, and the percentage of linear and circular monomer and dimer DNAs was determined. A peak of double-length, circular molecules (with the expected sedimentation constant of 38S) was found ahead of the 24S monomer peak. The double-length molecules had been purified 65-fold. As previously found for single-stranded DNA, the contour length of these molecules was strongly dependent upon ionic strength. Possible artifacts were ruled out, and it was shown that the double-length molecules arose from phage particles.     

Anatomy of Herpes Simplex Virus DNA. VIII. Properties of the Replicating DNA

This paper concerns the properties of herpes simplex virus 1 DNA replicating in HEp-2 and human embryonic lung cells. The results were as follows. (i) Only a small fraction of input viral DNA entered the replicative pool. The bulk of the input viral DNA cosedimented with marker viral DNA and did not appear to be degraded or dissociated into L and S components. (ii) Nascent DNA sedimented faster and banded at a higher density than that of mature viral DNA extracted from virions. Pulse-chase experiments indicated that nascent DNA acquires the sedimentation rate and buoyant density of viral DNA within 30 to 40 min after its synthesis. (iii) Electron microscopic studies indicated that the DNA extracted from cells replicating viral DNA and banding at the density of viral DNA contained: (a) linear, full-size molecules with internal gaps and single-stranded regions at termini; (b) molecules with lariats, consisting of a linear segment up to 2x the size of mature DNA and a ring ranging from 0.5 x 10(6) to 100 x 10(6) in molecular weight, showing continuous and discontinuous forks; (c) circular, double-stranded molecules, both full-size and multiples of 18 x 10(6) in molecular weight, but without forks or loops; (d) molecules showing "eye" and "D" loops at or near one end of the DNA; (e) large, tangled masses of DNA, similar to those observed for T4 and pseudorabies virus replicating DNAs, containing loops and continuous and discontinuous forks. The electron micrographs are consistent with the hypothesis that the single-stranded ends on the DNA anneal to form a hairpin, that the DNA synthesis is initiated at or near that end and proceeds bidirectionally to form a lariat, and that resulting progeny derived by semiconservative replication are "head-to-head" and "tail-to-tail" dimers.     

Single-stranded poly(deoxyguanylic acid) associates into double- and triple-stranded structures

Circular plasmid deoxyribonucleic acid (DNA), pBR322, was digested with the restriction endonuclease PstI to give full-length double-stranded DNA molecules, terminated by two self-complementary single-stranded sequences: (formula: see text). The protruding 3' termini were extended with dG by using calf thymus terminal deoxynucleotidyl transferase and dGTP, to form single-stranded tails of oligo(dG). At a length of about dG15, such tails become resistant to single strand specific endonuclease S1, and also cease to function as substrate (initiator) for the terminal deoxynucleotidyl transferase. This altered reactivity arises from association of the oligo(dG) tails into double- and triple-stranded structures, resulting in linear, circular, and branched polymers of the monomeric linear plasmid DNA. All these polymeric structures of the plasmid DNA are stable at room temperature, can be observed in the electron microscope, and can be separated from each other by agarose gel electrophoresis. At 60 degrees C or in 50% formamide, most of the oligo(dG) self-association can be reversed (melted), and the plasmid DNA is again found as the original linear monomer.     

Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques.

The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.     

Ultraviolet-Induced Cross-Links in the Deoxyribonucleic Acid of Single-Stranded Deoxyribonucleic Acid Viruses as a Probe of Deoxyribonucleic Acid Packaging

Deoxyribonucleic acid (DNA) from ultraviolet (UV)-irradiated phiX174 sediments in alkali at rates up to 1.7 times that of unirradiated phiX174 DNA and is observed as a condensed, cross-linked structure when examined in the electron microscope by the formamide spreading technique. This structure appears to result from multiple cross-links induced in the tightly coiled DNA contained within the spherical phiX174 capsid. In contrast, the DNA extracted after UV irradiation of the filamentous bacteriophage M13 is not strikingly altered in its sedimentation properties and appears by electron microscopy to be rod-shaped as a result of side-to-side association of the circular DNA. The differences in these UV-induced structures reflect the differences in the packaging of the single-stranded DNA in the two virions.