A study of the heterogeneous 37s ribonucleic acid induced by foot-and-mouth-disease virus

1. The 37s RNA induced in baby-hamster kidney cells by infection with foot-and-mouth-disease virus was examined on sucrose gradients and by filtration through Sepharose 4B. 2. The RNA sedimented faster (37s) and as a broader band than the 35s RNA from purified virus. 3. Treatment with deoxyribonuclease, Pronase or amylase did not alter the sedimentation profile of the 37s RNA. 4. Treatment of individual fractions of the RNA with phenol, dimethyl sulphoxide or methylCellosolve did not decrease the sedimentation rate of the faster-sedimenting molecules. 5. Sedimentation in sucrose gradients of different ionic strengths or containing EDTA had no effect on the heterogeneous nature of the profile. 6. On filtration through Sepharose 4B columns, the 37s virus-induced RNA was eluted before viral RNA. 7. Only 20% of the rapidly sedimenting RNA was incorporated into complete virus particles.     

Evidence for multiple vegetative DNA replication origins and alternative replication mechanisms of bovine papillomavirus type 1

By following up the chance detection in the electron microscope of a DNA replication intermediate within a preparation of bovine papillomavirus (BPV-1) DNA isolated from purified virus particles, information was obtained about the mechanism of BPV-1 genome replication during the final stages of virus multiplication in naturally infected bovine wart tissue. The structure of viral replication intermediates was investigated by electron microscopic analysis of viral DNA linearized by digestion with restriction endonucleases which cleave the circular BPV-1 chromosome at defined sites. Both Cairns and rolling circle-type molecules were identified. Furthermore, replication eyes were widely distributed within the viral genome, indicating that vegetative BPV-1 DNA replication origins are largely uncoupled from previously described plasmid maintenance sequence elements.     

Virus-Associated Nucleases: Evidence for Endonuclease and Exonuclease Activity in Rabbitpox and Vaccinia Viruses

The endonucleolytic action of a deoxyribonuclease activity in rabbitpox and vaccinia virus was established by change in sedimentation rate of denatured (3)H-lambda deoxyribonucleic acid substrate. The presence of two deoxyribonuclease activities in pox-virus is confirmed. Exo- and endonuclease activities are unmasked by treatment of purified virus with the detergent Nonidet P-40 and further enhanced by treatment of viral "cores" with trypsin.     

Selective degradation of integrated murine leukemia proviral DNA by deoxyribonucleases

The sensitivity to micrococcal nuclease and DNAase I of the integrated proviral DNA sequences in Swiss mouse cells infected with Moloney murine leukemia virus has been studied. Chromatin was separated into micrococcal nuclease-sensitive and -resistant regions, and the amount of proviral sequences in these DNA preparations was estimated by kinetic hybridization with single-stranded complementary DNA of Moloney murine leukemia virus. At least two thirds of the proviral DNA sequences were found in the open regions of chromatin, and only one third was resistant to nuclease. The proviral DNA sequences are even more sensitive to deoxyribonuclease I. When intact nuclei were treated with limited amounts of enzyme, only 5% of the nuclear DNA was digested, whereas 48% of the proviral DNA was degraded.The proviral DNA sequences in cells which do not produce virus are more resistant to nuclease digestion, as compared to virus producer cells. Thus the endogenous proviral sequences, in normal uninduced Swiss mouse cells, are randomly distributed between resistant and sensitive portions of chromatin when tested with either micrococcal nuclease or pancreatic deoxyribonuclease I. The effect of cell cycle synchronization on the accessibility of the proviral sequences to pancreatic deoxyribonuclease I was investigated with rat cells infected with Moloney murine leukemia virus. The amount of proviral DNA sensitive to pancreatic deoxyribonuclease I is higher in actively dividing cells than in cells arrested at Go phase, which produce only small amounts of virus.     

Mycoplasmal Deoxyribonuclease Activity in Virus-infected L-Cell Cultures

Cell-free extracts of Mycoplasma hominis and medium from 72-hr broth cultures had deoxyribonuclease activity like that of deoxyribonuclease I. Mg(++) stimulated activity, and the pH optimum was between 8.0 and 9.0. Double-stranded or heatdenatured deoxyribonucleic acid (DNA) served as a substrate, and oligonucleotides were produced. Cell-free extracts of L cells infected with M. hominis or M. hominis plus equine abortion virus (equine herpes virus, EAV) had greatly increased activity over that of extracts of L cells or of L cells infected with EAV alone. In the absence of M. hominis, however, extracts had little activity, most of which was in virus-infected cell cultures. Activity was found in the culture medium only in those systems in which M. hominis was present. It is concluded that M. hominis can contribute significant deoxyribonuclease activity to virus-infected as well as virusfree cell cultures. Perhaps the most interesting question arising concerns the ability of EAV, a DNA virus, to replicate successfully despite the presence of deoxyribonuclease activity at the site of replication (the nucleus).     

Physicochemical properties of fowlpox virus deoxyribonucleic acid and its anomalous infectious behavior

Randall, Charles C. (University of Mississippi, Jackson), Lanelle G. Gafford, Richard L. Soehner, and James M. Hyde. Physicochemical properties of fowlpox virus deoxyribonucleic acid and its anomalous infectious behavior. J. Bacteriol. 91:95-100. 1966.-Deoxyribonucleic acid (DNA) was extracted from fowlpox virus-infected tissue, purified inclusions, and purified virus by five variations of detergent and phenol methods. Phenol methods gave a poor yield, whereas detergent techniques extracted up to 78% of the DNA. The buoyant density was 1.695 g/ml, and the melting temperature in 7.2 m NaClO(4) was 39 C, both approximately equivalent to a guanine plus cytosine content of 35 moles per cent. Further proof of the double-stranded nature of the DNA was shown by the characteristic behavior toward deoxyribonuclease, formaldehyde, and heat. Infectious DNA was obtained by the various methods described, but this manifestation of biological activity was capricious and for unknown reasons was often not evident. The infectivity could not be related quantitatively to the amount of DNA employed. Furthermore, the infectious nature of fowlpox virus DNA was demonstrable only when the route of infection was the chorioallantoic membrane. In contrast, whole virus infected both membrane and chick skin with equal efficiency.     

Assessment of preferential cleavage of an actively transcribed retroviral hybrid gene in murine cells by deoxyribonuclease I, bleomycin, neocarzinostatin, or ionizing radiation

Preferential cleavage induced by bleomycin, neocarzinostatin, or ionizing radiation in a transcribed cellular gene was evaluated through comparisons with deoxyribonuclease I. The glucocorticoid-inducible LTL gene (a hybrid viral gene derived from mouse mammary tumor virus DNA) previously described [Zaret, K. S., & Yamamoto, K. R. (1984) Cell (Cambridge, Mass.) 38, 29-38] served as the specific DNA target. A Southern blot analysis was used to specifically assess cleavage of the LTL gene in nuclei isolated from cells either treated or untreated with the synthetic glucocorticoid dexamethasone. Hypersensitivity of the gene to bleomycin or neocarzinostatin, which paralleled deoxyribonuclease I hypersensitivity, was evident only in nuclei isolated from dexamethasone-treated cells. Like deoxyribonuclease I, sites of dexamethasone-inducible drug hypersensitivity were coincident with the binding region for the glucocorticoid receptor found within the regulatory sequences of the LTL gene. In contrast, no hypersensitivity to ionizing radiation was evident. Although bleomycin and neocarzinostatin showed qualitatively similar preferences for the transcribed LTL gene, quantitative evaluations of damage to total cellular DNA by filter elution showed that the relative specificity of bleomycin for the hypersensitive region was much less than that of either deoxyribonuclease I or neocarzinostatin.     

Properties of Maedi Nucleic Acid and the Presence of Ribonucleic Acid- and Deoxyribonucleic Acid-Dependent Deoxyribonucleic Acid Polymerase in the Virions

Maedi virus contains a ribonucleic acid (RNA) which can be resolved into three major components, namely, 62S, 33S, and 13S, by sucrose gradient centrifugation. The presence of RNA- and deoxyribonucleic acid (DNA)-dependent DNA polymerase in virions of maedi virus was demonstrated. The enzyme product could be converted into acid-soluble form by pancreatic deoxyribonuclease, but was resistant to digestion by pancreatic ribonuclease and to hydrolysis by NaOH.     

Demonstration of Infectious Deoxyribonucleic Acid in Transformed Cells I. Recovery of Simian Virus 40 from Yielder and Nonyielder Transformed Cells

Deoxyribonucleic acid (DNA) was extracted from virus-free simian virus 40 (SV40)-transformed hamster, mouse, and monkey cells and was inoculated into simian cells in the presence of diethylaminoethyl (DEAE)-dextran; infectious SV40 was recovered by using DNA from cell lines which fail to yield virus by the fusion technique as well as from cell lines which readily yield virus by fusion. The rescued virus was identified as SV40 by three methods: (i) neutralization of plaque formation by specific antiserum; (ii) induction of synthesis of viral-specific antigens detected by immunofluorescence; and (iii) presence of papovavirus particles seen by the electron microscope. Treatment of the transformed cell DNA with deoxyribonuclease or omission of the DEAE-dextran prevented the rescue of virus. Large amounts of transformed cell DNA were required (>10 mug/culture of 10(6) cells) to effect rescue of SV40 by passage through monkey cells. A linear response was obtained between the input of DNA with inocula between 10 and 45 mug of DNA/culture and the yield of SV40 recovered. Biological activity was demonstrable irregularly when the transformed cell DNA was assayed directly in the presence of DEAE-dextran. The DNA induced plaque formation in about 50% of the trials as well as the synthesis of SV40 tumor and viral antigens in rare simian cells. The infectious DNA appeared to be associated with cellular DNA. The infectivity was found in the pellet of precipitated DNA obtained by the Hirt technique and was inactivated by boiling for 15 min. These properties are characteristic of linear cellular DNA and not of free, circular SV40 DNA.     

Mature Form of the Deoxyribonucleic Acid from Chick Embryo Lethal Orphan Virus

The deoxyribonucleic acid (DNA) of chick embryo lethal orphan (CELO) virus, an oncogenic avian adenovirus, had a biphasic denaturation profile indicating intramolecular base composition heterogeneity. This was confirmed by shearing the DNA and centrifuging it to equilibrium in Cs(2)SO(4) in the presence of HgCl(2) when two bands were formed. No circular molecules formed when CELO virus DNA was annealed, although lambda DNA formed circles under the same conditions. No circular molecules were found by sedimentation or electron microscopy when the DNA was digested with exonuclease III and then annealed, but 30 to 40% of T7 DNA molecules became circular under similar conditions. The complementary strands of CELO virus DNA both appeared to be continuous, and, when CELO DNA was denatured and then annealed under appropriate conditions, all of the renatured molecules were linear. It is concluded that CELO virus DNA consists of a unique rather than permuted collection of linear molecules that lack exposed single-strand complementary ends or duplex terminal repetitions. These results are discussed in relation to the replication of viral DNA and the transformation of host cells.     

Linear, Single-Stranded Deoxyribonucleic Acid Isolated from Kilham Rat Virus

Kilham rat virus (KRV) was grown in a rat nephroma cell line and was purified by two isopycnic centrifugations in cesium chloride. The virus contains single-stranded deoxyribonucleic acid (DNA) with a molecular weight of approximately 1.6 x 10(6). The DNA was extracted from the virion by both phenol extraction and by 2% sodium dodecyl sulfate at 50 C. KRV DNA, extracted by both procedures, was observed in an electron microscope by using a cytochrome c or diethylaminoethyldextran monolayer. The DNA was also exposed to exonuclease I, an enzyme which hydrolyzes specifically linear, single-stranded DNA. Hydrolysis of 70 to 80% of the DNA was observed. Both the enzymatic and the electron microscope studies support the conclusion that extracted KRV DNA is a single-stranded, linear molecule. The length of the DNA was measured in the electron microscope and determined to be 1.505 +/- 0.206 mum.     

Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.     

The isolation of a concentrated and purified rotavirus preparation by using bentonite

The preparation obtained when cultivating virus SA-11 in the transplantable cell culture SPEV has been purified and concentrated by means of bentonite. The virus adsorption at acidic values of pH (4.0-4.5) and elution at alkaline values of pH (8.5) were the main stages of production of the concentrated and purified rotavirus preparation. The paper embraces data on the infective activity of the virus protein content, amount of the rotavirus antigen, results of the electron and immunoelectron microscopy, characterizing the method efficiency.     

Isolation and Partial Characterization of Different Defective DNA Molecules Derived from Polyoma Virus

Supercoiled DNA molecules purified from mouse cells infected with high-multiplicity-passaged polyoma virus has a broader size distribution and sediments more slowly than DNA derived from low-multiplicity-passaged virus. The shorter DNA molecules are predominately noninfectious. Virus populations containing distinct size classes of defective virus DNA were isolated by growing virus from single cells infected by a defective and nondefective helper virus (infectious center). This technique probably results in the cloning of defective virus particles. By applying the infectious center method to DNA from various fractions of sucrose gradients it has been possible to obtain shorter circular DNA molecules ranging in size from 50 to 95% of the unit-length polyoma DNA molecule. The shorter molecules in any one preparation are homogeneous in size. This class size is retained upon repeated passage of crude viral lysates at high multiplicity. Thus far, all the purified shorter DNA molecules tested appear to be noninfectious and largely resistant to cleavage by the R(1) restriction enzyme. Some of the purified defective molecules have been found to interfere with the production of infectious virus upon co-infection with unit-length infectious polyoma DNA.     

Effect of trypsin on mouse mammary tumor virus.

Undisrupted mouse mammary tumor virus (MuMTV) derived from the milk of of RIII mice has been analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy after treatment with insolubilized trypsin. No alterations were found in viral fine structure by either freeze-etch or negative-stain electron microscopy. No alterations were found in the ability of trypsinized virus to compete in a radioimmune assay for viral antigens. Infectivity experiments indicate no significant differences in the ability of treated virus to infect C57Bl mice. However, significant differences were observed in polypeptide composition. The intensely periodic acid-Schiff-positive band, gp140, was shown by galactose oxidase-borotritide labeling to be degraded into a fragment of 125,000 molecular weight. The major glycoprotein, gp55, was split into fragments of 36,000 and 23,000 molecular weight, both of which stained with periodic acid-Schiff stain. Gp68 was removed from the virus. Experiments with purified, iodinated gp55 showed that the trypsin-induced fragments of gp55 were immunologically active. We conclude that: (i) certain glycoproteins at the surface of MuMTV are accessible to an insoluble form of trypsin, (ii) the trypsin causes a nick in the polypeptide chain without affecting the configuration of the molecule; (iii) the nicked molecules remain bound to the virus; and (iv) the presence of these nicked molecules does not interfere with the biological or antigenic expression of virus function.     

浮游病毒的电镜观察

直接用戊二醛固定水样中的浮游病毒,通过超速离心使已固定的浮游病毒沉淀到覆有Formvar膜和碳支持膜的铜网上,经醋酸双氧铀染色后,利用透射电镜对湖水中的浮游病毒进行观察．结果可观察到球形、杆状和蝌蚪状等形态各异的浮游病毒颗粒及球形病毒的囊膜子粒、杆状病毒的核衣壳、具有不同尾部的蝌蚪状病毒等的精细超微结构．从而建立了一种简便、快捷和高效研究浮游病毒的电镜方法．     

Physico-chemical characteristics of the DNA of the nuclear polyhedrosis virus of the moth Porthetria dispar L

DNA preparations were obtained after dissolving the inclusion bodies, polyhedra virus particles, from the purified bundle virus of Porthetria dispar L. nuclear polyhedrosis. The DNA molecules in the preparations obtained are of different conformation and separate within the CsCl density gradient in the presence of ethidium bromide into supercoiled catenated and relaxed circular molecules (with the admixture of linear molecules). The circular DNA was studied by electron microscopy. The size of virus genome according to the data of reassociation kinetics of DNA is about 100 MD. Estimated on the basis of the values of buoyant density (p) and the melting temperature (Tmelt.) the content of guanine-cytosine pairs (GC pairs) in the viral DNA varies from 61 up to 65 mol%, and in the insect cell DNA--from 38 up to 40 mol%. The viral and cellular DNA are distinctly separated by centrifugation within the CsCl density gradient.     

Characterization of deoxyribonucleases induced by poxviruses

Increases in deoxyribonuclease activity assayed at alkaline pH can be observed in poxvirus-infected cells when native or denatured deoxyribonucleic acid (DNA) is used as substrate. The deoxyribonuclease assayable with native DNA as substrate, induced in HeLa cells by cowpoxvirus or vaccinia virus WR, can be separated from the corresponding enzyme present in normal cells by chromatography on diethylaminoethyl cellulose. In addition, the two enzymes induced in the virus-infected cells differ from each other in their chromatographic properties. The two induced enzymes have been further characterized with respect to properties of enzymatic reaction.     

The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies.

1. A homogeneous preparation of penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.     

A rapid NAD+-linked assay for microsomal uridine diphosphate glucuronyltransferase of rat liver and some observations on substrate specificity of the enzyme.

1. A new and rapid continuous assay of rat liver microsomal UDP-glucuronyltransferase (EC 2.4.1.17) has been developed. It is based on measurement of UDP production from UDP-glucuronate during the glucuronidation reaction; UDP production was continuously measured by coupling it to the conversion of NADH into NAD+ through pyruvate kinase and lactate dehydrogenase. This assay is independent of the acceptor substrate used; several findings confirm its applicability. 2. The glucuronidation rate of a series of phenol derivatives was determined with this assay, by using a Triton X-100-activated microsomal preparation as enzyme source. Conjugation of a series of nitrophenol derivatives was also investigated by the 'classical' assay (measurement of disappearance of the yellow colour of the nitrophenol during glucuronidation). The substrate with the highest conversion rate was 3-methyl-2-nitrophenol. 3. Both electron releasing and electron withdrawing ring substituents increased the glucuronidation rate of the phenol derivatives, as compared with phenol. 4. Lipid solubility seems important for determining the conversion rate: poorly lipid-soluble substrates were glucuronidated only at a low rate and high lipid solubility seems to be a prerequisite for high conversion rate. Glucuronidation of poorly lipid-soluble compounds may be limited by diffusion. 5. The consequences of these findings for the interpretation of studies on heterogeneity of the enzyme are discussed.