Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “MICROCOCCAL INFECTIONS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 991 records · Page 55Linked to original sources
PubMed1984

Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication.

Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin-promoter-enhancer region of SV40, when introduced into CV-1 or Cos-1 monkey cells by DEAE-dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin-specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non-replicating and post-replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein-DNA interactions during viral infection or upon cell differentiation.

Animals↗
PubMed1991

Replication of the simian virus 40 chromosome with purified proteins.

SV40 chromosomes prepared from infected CV-1 cells were replicated with the purified proteins of SV40 T antigen, HeLa DNA polymerase alpha-primase complex, single-stranded DNA-binding protein, and topoisomerases I and II, all of which have been shown to be essential for SV40 DNA replication in vitro. Replication started near the origin and proceeded bidirectionally. The maximum speed of replication fork movement was 200-300 nucleotides/min, which was similar to the rate of SV40 DNA replication with the same set of proteins. When replication products were digested with micrococcal nuclease, DNA fragments of 160-180 base pairs, which is the typical size of mononucleosomal DNA, were protected. This result indicates that replicated DNA was reconstructed into the nucleosome structure, complexed with parental histones.

Antigens, Polyomavirus Transforming↗
PubMed1971

Double-stranded poliovirus RNA inhibits initiation of protein synthesis by reticulocyte lysates.

Infection of HeLa cells with poliovirus results in an inhibition of host-cell protein synthesis. Cytoplasmic extracts from infected cells contian an activity that inhibits initiation of protein synthesis in vitro by rabbit reticulocyte lysates. This inhibitory activity has been purified and characterized, and is shown to reside in poliovirus double-stranded RNA. The intact double-stranded molecule is not essential for inhibitory activity.

Alanine↗
PubMed2005

[Capsid-targeted viral inactivation for dengue virus infection].

To explore the feasibility of capsid-targeted viral inactivation for dengue virus infection, a newly-discovered antiviral strategy, a mammalian cell line stably expressing staphylococcal nuclease fused to the capsid protein of dengue 2 virus was established and the effects on the production of infectious virus particles were examined. The results presented evidence that the enzymatically active staphylococcal nuclease fused to capsid protein could be incorporated into the nascent virions during wild virus assembly, resulting in degradation of viral genomic RNA and decrease in infectivity. Comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of SN, on the infectivity of progeny virions, nucleolytic activity of incorporated SN was responsible for the major antiviral effects. These results paved the road of developing capsid-targeted viral inactivation as a new antiviral strategy against dengue.

Aedes↗
PubMed1997

The assembly of the measles virus nucleoprotein into nucleocapsid-like particles is modulated by the phosphoprotein.

Measles virus nucleoprotein encoded from the vaccinia virus genome assembles into nucleocapsids similar in many respects to those observed during a natural measles virus infection. The influence of the measles virus phosphoprotein on nucleocapsid assembly has been studied using a vaccinia virus recombinant encoding both the nucleoprotein and the phosphoprotein. Infection of cells with the virus recombinant resulted in the formation of cytoplasmic inclusions in which the nucleoprotein and the phosphoprotein colocalized. Electron microscopic examination suggested that these inclusions contained characteristic nucleocapsid filaments. The buoyant density of nucleocapsids assembled in the presence of the phosphoprotein was found to be slightly higher than that of nucleocapsids assembled in its absence. Furthermore, the phosphoprotein partially inhibited the formation of nucleocapsids, a process which was extremely efficient when the nucleoprotein was expressed alone. Analysis of the nucleic acid content of nucleocapsids showed that they packaged heterologous RNA into a micrococcal nuclease-resistant form. These experiments demonstrate that the measles virus phosphoprotein regulates the efficiency with which the nucleoprotein assembles into nucleocapsids and the structural conformation they acquire.

Animals↗
PubMed1988

Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie.

Abnormal tubulofilamentous structures have been identified in electron micrographs of thin sections and negatively stained impression grids prepared from brains of animals with scrapie and other spongiform encephalopathies, and we showed that such tubules contain a core of filamentous structures resembling scrapie-associated fibrils (SAF). We treated impression grids from brains of scrapie-infected hamsters with several substances that bind to or cleave proteins and nucleic acids to see if they had any effect on the abnormal tubulofilamentous structures. Treatment with three proteolytic enzymes reduced the caliber of the tubules from about 50 nm to 30 nm; subsequent treatment of the 30-nm tubules with DNase I left many typical SAF as well as transitional forms in which twisted SAF emerged from tubules. DNase treatment of the original thicker tubules had no effect, and no SAF were seen on grids. Treatment of the 30-nm tubules with any of three other nucleases (micrococcal, mung bean, and BAL-31) also produced SAF. However, treatment with RNase A had no effect either on the original 50-nm tubules or on the 30-nm tubules produced by proteolysis. Detergent treatment of any of the preparations produced SAF. Treatment with ethidium bromide resulted in staining of the tubules that was inhibited by magnesium ions. The data suggest that the abnormal tubulofilamentous particles found in spongiform encephalopathies may consist of an outer cylinder of protein, an inner cylinder of DNA, and an innermost core of SAF.

Animals↗
PubMed2005

Association of bovine papillomavirus E2 protein with nuclear structures in vivo.

Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt.

Animals↗
PubMed1982

Minor coat protein composition and location of the A protein in bacteriophage f1 spheroids and I-forms.

The filamentous bacteriophage f1 can be transformed into a spherical particle (spheroid) or an intermediate shortened filament with a flared end (I-forms) by exposure to a chloroform-water interface at 22 or 4 degrees C, respectively. The protein composition of bacteriophage f1 spheroids and I-forms was examined by separating the proteins from the purified. [35S]cysteine-labeled particles by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Quantitation of the radioactivity on the gels showed that I-forms and spheroids contain the same complement of minor coat proteins as do untreated f1 phage. This composition is unchanged after removal of the DNA, either by digestion with micrococcal nuclease or by centrifugation of the particles through CsCl density gradients, indicating that none of the minor coat proteins is held in the particles solely through an interaction with the DNA. We also examined the location of the A protein in I-forms by decoration with ferritin-conjugated antibodies and examination under the electron microscope and found that the A protein is located specifically at the flared end of the I-form particle, through which the DNA is extruded and at which contraction into spheroids begins. The implications of these results with regard to the orientation of the DNA within the capsid and the process of infection are discussed.

Capsid↗
PubMed1976

Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA.

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.

Adenosine Triphosphate↗
PubMed1981

Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. II. Evidence for a strand displacement mechanism in plus-strand synthesis.

Analyses of the native DNA product of mellitin-activated avian retrovirus reverse transcription have revealed a unique structure. The vast majority of the molecules were linear, either 7.7 (genome) or 8.0 (extended genome) kilobases in length, and contained single-stranded DNA branches distributed throughout. These conclusions are based on electrophoretic properties of intact and restriction endonuclease-treated molecules before and after treatment with single-strand-specific nuclease S1. Preliminary data from linear viral DNA extracted from infected cells suggest that these molecules have a similar structure. The findings summarized in this report and those in the preceding paper indicated that the single-stranded branches are of positive polarity and are generated by a strand displacement mechanism. The existence of these branches suggests a role for strand displacement in replication and recombination.

Avian Leukosis Virus↗
PubMed2001

Characterization of intracellular reverse transcription complexes of human immunodeficiency virus type 1.

To examine the early events of the life cycle of human immunodeficiency virus type 1 (HIV-1), we analyzed the intracellular complexes mediating reverse transcription isolated from acutely infected cells. Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that two species of RTCs are formed but only one species is able to synthesize DNA. Most of the capsid, matrix, and reverse transcriptase (RT) proteins dissociate from the complex soon after cell infection, but Vpr remains associated with the RTC. The RTCs isolated 1, 4, and 7 h after infection are competent for reverse transcription in vitro, indicating that a small proportion of RT remains associated with them. HIV RTCs isolated early after infection have a sedimentation velocity of approximately 560S. Later, different species with a sedimentation velocity ranging from 350S to 100S appear. Nuclear-associated RTCs have a sedimentation velocity of 80S. Shortly after initiation of reverse transcription, the viral strong-stop DNA within the RTC is sensitive to nuclease digestion and becomes protected when reverse transcription is almost completed.

Blotting, Western↗
PubMed2004

Therapeutic effects of dengue 2 virus capsid protein and staphylococcal nuclease fusion protein on dengue-infected cell cultures.

Dengue infection poses a serious public health problem in most tropical and subtropical areas. No effective antiviral drugs or vaccines are currently available against dengue infection. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against dengue infection, we constructed a plasmid expressing a fusion protein consisting of staphylococcal nuclease (SN) fused to dengue 2 virus capsid protein (D2C), and investigated its effects on the production of infectious virions when introduced into BHK cells infected with dengue virus. The results indicated that D2C-SN can be expressed and tolerated in this mammalian cell culture. The enzymatically active SN moiety was incorporated into nascent virions during the process of viral assembly. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of wild-type SN, on the infectivity of progeny virions, we clearly demonstrated that nucleolytic activity was the major antiviral mechanism. Expression of D2C-SN fusion protein as a therapeutic agent resulted in a reduction in infectious titers of 12- to 60-fold. Therefore, dengue virus may be particularly vulnerable to a CTVI therapeutic approach.

Animals↗
PubMed1980

Synthesis and processing of Semliki Forest virus-specific nonstructural proteins in vivo and in vitro.

A large short-lived virus-specific nonstructural protein with an apparent molecular weight of about 250000 (nsp250) has been isolated from cells infected with the temperature-sensitive mutants ts-4 and ts-6 of the Semliki Forest virus. nsp250 contained all peptides characteristic of the two previously identified nonstructural precursor proteins, nsp155 and nsp135, as revealed by limited proteolysis with Staphylococcus aureus V8 protease. Thus nsp250 is probably the translational product of the 5' two-thirds of the 42-S RNA genome which codes for the virus-specific nonstructural proteins. A second viral nonstructural precursor protein, nsp220, was also characterized by peptide mapping. This protein contained all the peptides of nsp155, and several but not all of the peptides of nsp135. Some peptides were demonstrated which possibly are derived from ns60, the only nonstructural protein not yet isolated. Small amounts of proteins with identical mobility to nsp250 and nsp220 were synthesized at 38 degrees C in micrococcal-nuclease-treated rabbit reticulocyte lysate in response to virion 42-S RNA from the ts-6 mutant. The product of the wild-type 42-S RNA in vitro contained, in addition to nsp220 and nsp155, polypeptides which comigrated with ns86, ns72 and ns70, indicating processing of the translational product. The authenticity of nsp220, nsp155 and ns70 synthesized in vitro was confirmed by limited proteolysis with V8 protease.

Animals↗
PubMed2004

On the mechanism of constitutive Pdr1 activator-mediated PDR5 transcription in Saccharomyces cerevisiae: evidence for enhanced recruitment of coactivators and altered nucleosome structures.

Drug resistance as a result of overexpression of drug transporter genes presents a major obstacle in the treatment of cancers and infections. The molecular mechanisms underlying transcriptional up-regulation of drug transporter genes remains elusive. Employing Saccharomyces cerevisiae as a model, we analyzed here transcriptional regulation of the drug transporter gene PDR5 in a drug-resistant pdr1-3 strain. This mutant bears a gain-of-function mutation in PDR1, which encodes a transcriptional activator for PDR5. Similar to the well studied model gene GAL1, we provide evidence showing that PDR5 belongs to a group of genes whose transcription requires the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex. We also show that the drugindependent PDR5 transcription is associated with enhanced promoter occupancy of coactivator complexes, including SAGA, Mediator, chromatin remodeling SWI/SNF complex, and TATA-binding protein. Analyzed by chromatin immunoprecipitations, loss of contacts between histones and DNA occurs at both promoter and coding sequences of PDR5. Consistently, micrococcal nuclease susceptibility analysis revealed altered chromatin structure at the promoter and coding sequences of PDR5. Our data provide molecular description of the changes associated with constitutive PDR5 transcription, and reveal the molecular mechanism underlying drug-independent transcriptional up-regulation of PDR5.

ATP-Binding Cassette Transporters↗
PubMed1987

Pre-mRNA splicing and the nuclear matrix.

We examined the relationship between pre-mRNA splicing and the nuclear matrix by using an in vivo system that we have developed. Plasmids containing the inducible herpesvirus tk gene promoter linked to an intron-containing segment of the rabbit beta-globin gene were transfected into HeLa cells, and then the promoter was transactivated by infection with a TK- virus. Northern analysis revealed that the globin pre-mRNA and all its splicing intermediates and products are associated with the nuclear matrix prepared from such transfected cells. When the nuclear matrix was incubated with a HeLa cell in vitro splicing extract in the presence of ATP, the amount of matrix-associated precursor progressively decreased without a temporal lag in the reaction, with a corresponding increase in free intron lariat. Thus, most of the events of the splicing process (endonucleolytic cuts and branching) occur in this in vitro complementation reaction. However, ligation of exons cannot be monitored in this system because of the abundance of preexisting mature mRNA. Since the matrix is not a self-splicing entity, whereas the in vitro splicing system cannot process efficiently deproteinized matrix RNA, we conclude from our in vitro complementation results (which can be reproduced by using micrococcal nuclease-treated splicing extract) that the nuclear matrix preparation retains parts of preassembled ribonucleoprotein complexes that have the potential to function when supplemented with soluble factors (presumably other than most of the small nuclear ribonucleoproteins known to participate in splicing) present in the HeLa cell extract.

Animals↗
PubMed1977

Bacteriophage T4D head morphogenesis. VIII. DNA-protein associations in intermediate head structures that accumulate in gene 49--mutant-infected cells.

We have utilized the gene 49(-) mutant-infected cells of bacteriophage T4D to accumulate large numbers of nucleic acid-protein intermediate head structures. These heads were used as substrates for experiments in the investigations of the mechanism of DNA packaging. Specifically, we have examined: (i) the susceptibility of the DNA in these structures to digestion by a variety of nucleases after a series of increasing temperature pulses from 25 to 100 degrees C, (ii) the physicochemical characteristics of the DNA inside these heads, and (iii) the mechanism by which proteins are displaced from the interior of the head after treatment with basic proteins. We isolated DNA from these gene 49(-) heads by use of gradient centrifugation procedures. The DNA had a molecular weight of 8 x 10(6) and a density of 1.697 +/- 0.005 g/cm(3), and it contained a short resistant fraction (SRF) which, when associated with the gene 49(-) heads, exhibited AT-protected regions that were not susceptible to micrococcal nuclease digestion. Such a fraction may contain pieces which are important in the initial association of the DNA with the prohead. Exposure of the gene 49(-) intermediate capsid structures to basic proteins, such as bovine trypsin inhibitor, lysozyme, and l-polylysine-70, caused a displacement of an amorphous-appearing structure which may be a complex of the gene 49(-) DNA and interior components of the capsid (e.g., internal proteins, polyamines). Our general conclusion is that in the gene 49(-) intermediate head structures which are only partly filled with DNA, this DNA is held inside the head by strong electrostatic linkages with interior polypeptides and polyamines.

Capsid↗
PubMed1981

Replication of parvoviral DNA. I. Characterization of a nuclear lysate system.

We have developed a nuclear lysate system from infected, synchronized cells capable of synthesizing unit-length parvoviral DNA in vitro. It was necessary to supplement the nuclear lysates with the polyamines, spermidine and spermine, to prevent degradation of template and product DNAs. In this system RF, RI, and single-stranded progeny DNAs were synthesized. Label incorporated in viral RF DNA in vivo appeared first in RI DNA and then in single-stranded DNA during incubation in vitro. By sedimentation the product DNAs were identical to those found in infected cells. Their viral identity was confirmed by hybridization. The addition of ribonucleotides, RNase, or alpha-amanitin did not affect parvoviral DNA synthesis in this system. The results with the specific inhibitors of mammalian DNA polymerases, aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'-triphosphate indicated that DNA polymerase alpha was required for synthesis of parvoviral DNA in the nuclear lysates. This requirement was confirmed by experiments with antibody to bovine DNA polymerase alpha.

Animals↗
PubMed1996

Synthesis of potato virus X RNAs by membrane-containing extracts.

Membrane-containing extracts isolated from tobacco plants infected with the plus-strand RNA virus, potato virus X (PVX), supported synthesis of four major, high-molecular-weight PVX RNA products (R1 to R4). Nuclease digestion and hybridization studies indicated that R1 and R2 are a mixture of partially single-stranded replicative intermediates and double-stranded replicative forms. R3 and R4 are double-stranded products containing sequences typical of the two major PVX subgenomic RNAs. The newly synthesized RNAs were demonstrated to have predominantly plus-strand polarity. Synthesis of these products was remarkably stable in the presence of ionic detergents.

Endonucleases↗