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At least 973 records · Page 54Linked to original sources
PubMed1978

Chromatin assembly in isolated mammalian nuclei.

Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [3H]thymidine and then incubated in the presence of [alpha-32P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease of DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs). (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest. (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions.

Cell Line↗
PubMed1990

Assembly of nascent DNA into nucleosome structures in simian virus 40 chromosomes by HeLa cell extract.

A soluble system was developed that could support DNA replication in simian virus 40 (SV40) chromosomes. DNA synthesis in this system required the presence of purified SV40 large tumor antigen, SV40 chromosomes prepared from virus-infected monkey cells, a crude extract from HeLa cells, and several low-molecular-weight components. In comparison to the replication of purified SV40 form I DNA, the rate of DNA synthesis was 15 to 20% in this system. DNA synthesis started near the replication origin of SV40 and proceeded bidirectionally in a semiconservative manner. Micrococcal nuclease digestion experiments revealed that the replicated DNA produced in this system became organized into a regularly spaced array of nucleosome core particles when an appropriate amount of purified HeLa core histones was added to the reaction mixture. SV40 form I DNA replicating under the same conditions was also assembled into nucleosomes, which were arranged in a rather dispersed manner and formed an aberrant chromatin structure.

Cell Line↗
PubMed1992

Incorporation of the carbocyclic analog of 2'-deoxyguanosine into the DNA of herpes simplex virus and of HEp-2 cells infected with herpes simplex virus.

The carbocyclic analog of 2'-deoxyguanosine (CdG) is active against herpes simplex virus (HSV), human cytomegalovirus, and human hepatitis-B virus. In order to understand the mechanism of action of this compound against HSV, we have evaluated (a) the incorporation of [3H]CdG into viral and host DNA in HEp-2 cells infected with HSV and (b) the interaction of the 5'-triphosphate of CdG (CdG-TP) with the HSV DNA polymerase and human DNA polymerases alpha, beta, and gamma (EC 2.7.7.7). Incubation of HSV-1-infected HEp-2 cells with [3H]CdG resulted in the incorporation of CdG into both the HSV and the host cell DNA. These results indicated that CdG-TP was used as a substrate for HSV DNA polymerase and for at least one of the cellular DNA polymerases. Degradation of both viral and host DNA with micrococcal nuclease and spleen phosphodiesterase indicated that CdG was incorporated primarily into internal positions in both DNAs. The viral DNA containing CdG sedimented in neutral and alkaline sucrose gradients in the same way as did viral DNA labeled with [3H]thymidine, indicating that the HSV DNA containing CdG was similar in size to untreated HSV DNA. CdG-TP was a competitive inhibitor of the incorporation of dGTP into DNA by the HSV DNA polymerase (Ki of 0.35 microM) and the human DNA polymerase alpha (Ki of 1 microM). CdG-TP was not a potent inhibitor of either DNA polymerase beta or gamma. Using DNA-sequencing technology, CdG-TP was found to be an efficient substrate for HSV DNA polymerase. Incorporation of CdG monophosphate (CdG-MP) into the DNA by HSV DNA polymerase did not interfere with subsequent chain extension. These results suggested that the antiviral activity of CdG was due to its incorporation into the DNA and subsequent disruption of viral functions. In contrast, CdG-TP was not as good as dGTP as a substrate for DNA synthesis by DNA polymerase alpha, and incorporation of CdG-MP by DNA polymerase alpha inhibited further DNA chain elongation.

Base Sequence↗
PubMed1983

(2'-5')Oligoadenylate in rat liver: modulation after partial hepatectomy.

(2'-5')Oligoadenylate synthetase [(2'-5')A synthetase], which synthesizes a series of oligoadenylates ppp-(A2'p)n5'A [collectively referred to as (2'-5')A], has been described previously in rat liver cells, where its concentration varied with the growth status of this organ--i.e., it decreased during the early phase of rat liver regeneration after partial hepatectomy. Because double-stranded RNA, the only known activator of this enzyme, has been detected in rat liver nuclei, (2'-5')A synthesis could occur in this tissue in vivo. Analysis of rat liver cell extract after HPLC by the endonuclease-based radiobinding assay revealed several components with retention times similar to (2'-5')A trimer- and tetramer-like material. A further characterization of these compounds by their susceptibility to alkaline phosphatase and snake venom phosphodiesterase, their resistance to micrococcal nuclease, and their ability to activate an endonuclease indicated the natural occurrence of oligonucleotides indistinguishable from authentic (2'-5')A in rat liver cells. Using the combination of the radiobinding assay and a simplified (2'-5')A extraction procedure that does not involve HPLC, we further show that the early drop of (2'-5')A synthetase activity during rat liver regeneration was accompanied by a similar decrease in intracellular (2'-5')A concentration. The three characteristic phases of the (2'-5')A synthetase kinetics during the first 40 hr of liver regeneration were mimicked by the kinetics of the synthesis of the (2'-5')A oligonucleotides themselves: between 6 and 20 hr after hepatectomy, there was a sharp decrease in (2'-5')A concentration; between 20 and 24 hr, the concentration of (2'-5')A reached a minimum; at 36 hr or after the first wave of DNA synthesis (the major event of liver regeneration), the (2'-5')A concentration returned to normal. In this characterization of the (2'-5')A oligonucleotide family in a functional tissue of an animal that had not been previously treated with interferon or infected with virus, the data are compatible with a physiological role of the (2'-5')A system acting as an intracellular component of the regulatory mechanisms leading to cell proliferation or differentiation.

Adenine Nucleotides↗
PubMed1985

Template-dependent RNA polymerase from black beetle virus-infected Drosophila melanogaster cells.

Infection of cultured cells of Drosophila melanogaster with black beetle virus (BBV) induces an RNA polymerase that is bound to cellular particulate material in a complex with a template RNA. We have solubilized the polymerase by treatment of the relevant particulates with detergents such as dodecyl-beta-D-maltoside. The polymerase activity was made dependent upon exogenous RNA by destruction of the endogenous template RNA with micrococcal nuclease. Addition of BBV RNA1 or RNA2 induced synthesis of full-length negative-strand RNA isolated as a double-stranded complex with the added RNA. Newly synthesized plus strands were also detected in the RNA2 complexes. Certain other viral RNAs also induced synthesis of their negative strands.

Animals↗
PubMed1981

[Translation of chicken interferon messenger in a cell-free protein synthesis system and in a cell culture].

A highly effective cell-free system for protein synthesis was obtained from rabbit reticulocytes and for the first time used for synthesis of biologically active chicken interferon. The optimal conditions for translation of its mRNA were developed. The translation efficacy in the cell-free system was 10-50 times higher than that in the culture of heterologous cells. The higher the purity level of RNA, the higher the translation level. With respect to poly (A+) RNA sedimenting in the sucrose gradient 9S the efficacy reached 2560 units per 1 microgram of RNA. By the content of poly (A), sequences and rate of the sedimentation, mRNA of the chicken interferon was similar to that of the human fibroblast cell interferon. The possible translation of mRNA of the chicken interferon at low concentrations of exogenic potassium ions in the cell-free system is explained by production of interferon in infected cells where the concentration of the intracellular potassium significantly decreases which is indicative of the mRNA interferon similarity with virus templates. It was found that only albino New Zealand rabbits, but also chinchilla may be used for preparation of the cell-free protein synthesizing system. Various exogenic templates in the mRNA-dependent cell-free system prepared from reticulocyte nonfractionated lysate by treatment with micrococcal nuclease stimulated the protein synthesis by 7-15 times.

Animals↗
PubMed1983

Discriminatory inhibition of protein synthesis in cell-free systems by vaccinia virus transcripts.

The effect of vaccinia virus early transcripts on cellular (globin, HeLa, Chinese hamster ovary) and viral (vaccinia, encephalomyocarditis) mRNA function was studied in reticulocyte and wheat germ cell-free protein-synthesizing systems. Vaccinia virus transcripts of two size classes (8-10 S and 4-7 S), generated in vitro by viral cores, inhibited function of cellular and encephalomyocarditis virus mRNA but not that of vaccinia virus in reticulocyte lysate systems. Mild alkaline hydrolysis or micrococcal nuclease treatment of vaccinia virus in vitro transcripts resulted in a loss of their ability to inhibit protein synthesis directed by HeLa cell RNA. Vaccinia virus in vitro transcripts also selectively inhibited HeLa cell protein synthesis in wheat germ systems, suggesting that double-stranded RNA is not involved in this inhibition of protein synthesis. The addition, to the reticulocyte translating system, of cytoplasmic RNA obtained from infected cells in conjunction with cellular mRNA (globin, HeLa) resulted in the inhibition of synthesis of the globin or HeLa polypeptides with little or no effect on the translation of the vaccinia virus proteins. RNA extracted from vaccinia virions inhibited cellular but not vaccinia virus mRNA function when added to the reticulocyte lysate systems with uninfected or infected HeLa cell cytoplasmic RNA.

Animals↗
PubMed1982

Intracellular DNA of the parvovirus minute virus of mice is organized in a minichromosome structure.

Minute virus of mice (MVM) nucleoprotein complexes were leached from infected cell nuclei in the presence of a hypotonic buffer. Detailed biochemical analyses performed on the extracted complexes revealed nucleoprotein complexes sedimenting together with virions at 110S and defective particles sedimenting at 50S. In contrast to the virions, the nucleoprotein complexes were found to be sensitive to treatment with DNase, Sarkosyl, and heparin. They were found to be composed of replicative forms of MVM DNA and cellular histones. After extensive micrococcal nuclease digestion performed on purified nucleoprotein complexes, a viral nucleosomes core containing a DNA segment of about 140 base pairs in length was identified. These complexes when visualized by electron microscopy revealed the existence of beaded structures (minichromosomes) having 26 and 52 beads per monomer and dimer molecules, respectively. We suggest that the organization of the intracellular viral DNA in a minichromosome structure is an essential step in the virus growth cycle.

Chromosomes↗
PubMed1984

Dispersive segregation of nucleosomes during replication of simian virus 40 chromosomes.

The distribution of preformed ("old") histone octamers between the two arms of DNA replication forks was analyzed in simian virus 40(SV40)-infected cells following treatment with cycloheximide to prevent nucleosome assembly from nascent histones. Viral chromatin synthesized in the presence of cycloheximide was shown to be deficient in nucleosomes. Replicating SV40 DNA (wild-type 800 and capsid assembly mutant, tsB11) was radiolabeled in either intact cells or nuclear extracts supplemented with cytosol. Nascent nucleosomal monomers were then released by extensive digestion of isolated nuclei, nuclear extracts or isolated viral chromosomes with micrococcal nuclease. The labeled nucleosomal DNA was purified and found to hybridize to both strands of SV40 DNA restriction fragments taken from each side of the origin of DNA replication, whereas Okazaki fragments hybridized only to the strand representing the retrograde DNA template. In addition, isolated, replicating SV40 chromosomes were digested with two strand-specific exonucleases that excised nascent DNA from either the forward or the retrograde side of replication forks. Pretreatment of cells with cycloheximide did not result in an excess of prenucleosomal DNA on either side of replication forks, but did increase the amount of internucleosomal DNA. These data are consistent with a dispersive model for nucleosome segregation in which "old" histone octamers are distributed to both arms of DNA replication forks.

Centrifugation, Density Gradient↗
PubMed1988

Structure and protein composition of the rotavirus replicase particle.

To understand the role of viral proteins in the replication of rotavirus RNA, we have characterized the structure of subviral particles (SVPs) that synthesize double-stranded RNA (DS RNA). Pulse-labeling of newly made RNA in infected cells showed that rotavirus DS RNA was synthesized either in single-shell (SS)-like particles or in precursor particles that rapidly mature into SS particles. Experiments using a cell-free system demonstrated that most replicase particles containing newly made DS RNA were of greater density in CsCl than single-shelled (SS) particles. However, this was partly due to the presence of single-stranded RNAs as the treatment of replicase particles with micrococcal nuclease reduced their density to between core particles and SS particles. Electrophoretic analysis indicated that replicase particles, purified by centrifugation on CsCl and glycerol gradients, were similar to SS particles, containing the structural proteins VP1, VP2, and VP6. Rotavirus replicase particles were also found to contain the nonstructural proteins NS34 and NS35 and possible host components. The presence of VP6 in enzymatically active replicase particles suggests that, like transcription, this protein may be required for rotavirus RNA replication.

Capsid↗
PubMed1982

Evidence that vesicular stomatitis virus produces double-stranded RNA that inhibits protein synthesis in a reticulocyte lysate.

Cell-free protein synthesis by reticulocyte lysates was inhibited by a polyadenylated RNA fraction extracted from HeLa cells infected with vesicular stomatitis virus (VSV) but not by polyadenylated RNA from mock-infected HeLa cells. A similar inhibitor of cell-free protein synthesis was found in a polyadenylated fraction of RNA transcribed in vitro by VSV but not in untranscribed nucleocapsids. Fractionation of the VSV transcription product showed that the translation inhibitor segregated with nucleocapsids containing newly transcribed polyadenylated or non-polyadenylated RNA, as determined by oligodeoxythymidylate-cellulose chromatography. The inhibitors present in VSV-infected HeLa cells and in VSV in vitro transcripts both appeared to be double-stranded RNA, as judged by the characteristics for inhibition of reticulocyte cell-free protein synthesis described by Hunter et al. (J. Biol. Chem. 250:409-417, 1975). The double-stranded nature of the VSV RNA inhibitor was supported by the finding that the translational inhibitory effect was inactivated by melting the inhibitor in the absence of salt and by micrococcal nuclease.

Cell-Free System↗
PubMed1958

Antibiotic resistant staphylococci.

An antibiotic resistant staphylococcus with bacteriophage pattern 52/42B/80/81* is frequently responsible for infectious outbreaks in the newborn nursery. Some time after an outbreak had occurred in the University of California's hospital nursery, family members of the infants were found to be infected with this strain. Two families were studied in detail. In one of them, infection developed in six of the seven members within eight months after the infant's arrival. In the other, half of the family members had recurrent infections during a 13-month period.Infants who left the nursery as asymptomatic carriers were found as likely to transmit the infectious strain as those with clinical infection. Considerable time sometimes elapsed before infection developed in either the infant or the family members. In one instance the first familial infection occurred six months after the infant had left the nursery as an asymptomatic carrier. Newborn infants are quite likely to disseminate antibiotic resistant staphylococci which they may acquire from a hospital nursery. Infections developing among persons in contact with a young infant must be treated with the possibility of a resistant hospital staphylococcus in mind.

Anti-Bacterial Agents↗
PubMed1995

Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus.

A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl beta-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.

Amino Acid Sequence↗
PubMed2006

Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound.

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.

Cell Membrane↗
PubMed2004

ORC, MCM, and histone hyperacetylation at the Kaposi's sarcoma-associated herpesvirus latent replication origin.

The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells. The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an approximately 150-bp region within the viral terminal repeats (TR). We have used chromatin immunoprecipitation assays to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells. The latent replication origin within the TR was also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and was enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome. Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, bound TR in late-G(1)/S-arrested cells, which coincided with the loss of histone H3 K4 methylation. Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G(1)/S phase. ORC binding to TR was LANA dependent when reconstituted in transfected plasmids. DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bound TR specifically and identified the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) as a potential TR binding protein. Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids. These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication. These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats.

Acetylation↗
PubMed1994

Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell.

Hepatitis B virus (HBV) nucleoprotein complexes were isolated from nuclei of the human hepatoblastoma cell line HepG2.2.15. Under conditions of physiological ionic strength, the complexes sedimented at a rate corresponding to about 82 S. They contained viral DNA, histone, and nonhistone proteins. For DNA a circular, covalently closed structure was shown both by CsCl gradient centrifugation and electron microscopy. Spread preparations revealed the typical "beads-on-a-string" appearance of nucleosomally organized DNA. The average number of nucleosomes was 18, resulting in a biochemical repeat unit of HBV chromatin of approximately 180 base pairs of DNA. This value was confirmed by experiments analyzing the structure of the HBV chromatin by the use of micrococcal nuclease. Electron microscopy demonstrated that exposure to high ionic strength conditions resulted in removal of nucleosomes from the complexes, but also revealed proteinaceous structures remaining bound to viral DNA molecules. The nature of these residual proteins is discussed. Since native nucleoprotein complexes could be precipitated with HBV-core antibodies, core protein appeared to be one of the nonhistone proteins.

Centrifugation, Density Gradient↗
PubMed1988

In vitro assembly of a functional nucleocapsid from the negative-stranded genome RNA of a defective interfering particle of vesicular stomatitis virus.

The template for transcription and replication of negative-stranded RNA viruses is a ribonucleoprotein structure, the nucleocapsid. We have developed a system that supports assembly of the negative-stranded RNA genome of a defective interfering (DI) particle of vesicular stomatitis virus (VSV) into a nucleocapsid in vitro. This system uses extracts from wild-type VSV-infected cells as a source of proteins to encapsidate the RNA. In vitro assembled nucleocapsids were compared to in vivo-derived nucleocapsids by the following characteristics: nuclease resistance of the encapsidated RNA, CsCl density banding of labeled RNA in a position coincident with nucleocapsids, correct sedimentation rate in sucrose gradients, the presence of the nucleocapsid protein on the nucleocapsids, and the infectivity of the in vitro assembled nucleocapsids. We conclude that the system we present is capable of assembling the isolated genome of a rhabdovirus DI particle into nucleocapsids indistinguishable from those produced during the course of intracellular DI replication.

Animals↗