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At least 937 records · Page 52Linked to original sources
PubMed1982

Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP.

Incubation of extracts of phi 29-infected Bacillus subtilis with [alpha-32P]dATP produced a labeled protein having the electrophoretic mobility of p3, the 5'-terminal protein of phi 29 DNA. The reaction product was resistant to treatment with micrococcal nuclease, phosphatase, and RNases A and T1 and sensitive to proteinase K. Incubation of the 32P-labeled protein with piperidine under conditions in which the phi 29 DNA-protein p3 linkage is hydrolyzed released 5'-dAMP. The reaction with [alpha-32P]dATP was strongly inhibited by anti-p3 serum and required the preence of phi 29 DNA-protein p3 complex; no reaction took place with proteinase K-treated phi29 DNA. These results, together with those of acid hydrolysis and partial proteolysis, indicated that a covalent complex between protein p3 and 5'-dAMP is formed in vitro. The initiation complex (protein p3-dAMP) formed in the presence of 0.5 microM [alpha-32P]dATP can be elongated by addition of 40 microM dNTPs. Treatment with piperidine of the product elongated in the presence of 2',3'-dideoxycytidine 5'-triphosphate released the expected oligonucleotides, 9 and 12 bases long, taking into account the sequence at the left and right DNA ends, respectively.

Bacillus subtilis↗
PubMed1978

Photochemical addition of the cross-linking reagent 4,5', 8-trimethylpsoralen (trioxaslen) to intracellular and viral simian virus 40 DNA-histone complexes.

We demonstrated here that 4,5', 8-trimethylpsoralen (trioxsalen) is a valuable probe for the structure of SV40 DNA-histone complexes. Trioxsalen readily penetrated intact cells and, in the presence of 340- to 380-nm light, covalently cross-linked DNA preferentially at the sites available for micrococcal nuclease digestion. Histograms of the lengths of the regions of SV40 DNA protected from cross-linking, as visualized by electron microscopy, indicated a repeating pattern of base pairs in DNA from both infected cells and virus particles. The ability of the trioxsalen probe to act in vivo and to map the location of protected regions may provide a powerful tool for analyzing the role of nucleosomes in the structure of the virus particle and in intracellular complexes such as transcription templates and replication intermediates.

DNA, Viral↗
PubMed1991

Properties of human parainfluenza virus type 3 RNA polymerase/replicase activity in vitro: consensus with other negative-stranded RNA viruses.

A cell-free system supporting transcription, replication, and nucleocapsid assembly of the genome RNA of human parainfluenza virus type 3 (HPF3) is described. Cytoplasmic extracts from infected CV-1 or BHK cells catalyzed the transcription of the entire HPF3 genome, the replication of genome RNA, and the assembly of this RNA into nucleocapsidlike structures. Newly replicated RNA was resistant to micrococcal nuclease digestion and was stable in CsCl gradients, exhibiting the density of authentic HPF3 nucleocapsids. After fractionation of the extracts, the nucleocapsid-containing pellet fraction synthesized viral mRNAs. Reconstitution with the soluble protein fraction was necessary for genome RNA replication and nucleocapsid assembly.

Animals↗
PubMed2003

Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay.

A screening method for methicillin-resistant Staphylococcus aureus (MRSA) by using selective broth and real-time PCR (broth-PCR) was developed and evaluated. The samples (n = 304) were cultured in the broth overnight, followed by nuc gene detection by real-time PCR. nuc-negative samples were further checked for the presence of nuc amplification inhibitors by a PCR internal inhibitor assay. nuc-positive samples and nuc-negative samples with PCR inhibitors were cultured onto plates and processed further. The diagnostic values for this MRSA screening method were 93.3% sensitivity, 89.6% specificity, 31.8% positive predictive value, and 99.6% negative predictive value. The application of the broth-PCR method will be able to report most of the negative samples (258 of 289 [89.3%]) on the next morning and can save as much as 84.9% (258 of 304) of the labor and cost spent on processing the nuc-negative specimens on plates. In the study, all the samples were processed in parallel by the broth enrichment method and the plating method for comparison. To identify MRSA, the isolated oxacillin-resistant S. aureus strains were tested by a duplex real-time PCR targeting the mecA gene and the nuc gene. A collection of MRSA, methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, and methicillin-susceptible Staphylococcus epidermidis strains and a panel of standard strains of 11 bacterial species other than S. aureus were also tested by this method, which was proved to be a valuable tool for MRSA identification in a routine microbiological laboratory.

Bacterial Proteins↗
PubMed2003

Chromatin remodeling of the Kaposi's sarcoma-associated herpesvirus ORF50 promoter correlates with reactivation from latency.

The switch from latent to lytic infection of Kaposi's sarcoma-associated herpesvirus is initiated by the immediate early transcriptional activator protein Rta/open reading frame 50 (ORF50). We examined the transcriptional regulation of the ORF50 core promoter in response to lytic cycle stimulation. We show that the ORF50 promoter is highly responsive to sodium butyrate (NaB) and trichostatin A (TSA), two chemicals known to inhibit histone deacetylases. The NaB and TSA responsive element was mapped to a 70-bp minimal promoter containing an essential GC box that binds Sp1/Sp3 in vitro and in vivo. Micrococcal nuclease mapping studies revealed that a nucleosome is positioned over the transcriptional initiation and the Sp1/3 binding sites. Stimulation with NaB or TSA increased histone acetylation and restriction enzyme accessibility of the ORF50 promoter transcription initiation site. Chromatin immunoprecipitation assay was used to demonstrate that the ORF50 promoter is associated with several different histone deacetylase proteins (including HDAC1, 5, and 7) in latently infected cells. NaB treatment led to the rapid association of Ini1/Snf5, a component of the Swi/Snf family of chromatin remodeling proteins, with the ORF50 promoter. Ectopic expression of the CREB-binding protein (CBP) histone acetyltransferase (HAT) stimulated plasmid-based ORF50 transcription in a HAT-dependent manner, suggesting that CBP recruitment to the ORF50 promoter can be an initiating event for transcription and viral reactivation. Together, these results suggest that remodeling of a stably positioned nucleosome at the transcriptional initiation site of ORF50 is a regulatory step in the transition from latent to lytic infection.

Base Sequence↗
PubMed2003

Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments.

Flavivirus infection causes extensive proliferation and reorganization of host cell membranes to form specialized structures called convoluted membranes/paracrystalline arrays and vesicle packets (VP), the latter of which is believed to harbor flaviviral replication complexes. Using detergents and trypsin and micrococcal nuclease, we provide for the first time biochemical evidence for a double membrane compartment that encloses the replicative form (RF) RNA of the three pathogenic flaviviruses West Nile, Japanese encephalitis, and dengue viruses. The bounding membrane enclosing the VP was readily solubilized with nonionic detergents, rendering the catalytic amounts of enzymatically active protein component(s) of the replicase machinery partially sensitive to trypsin but allowing limited access for nucleases only to the vRNA and single-stranded tails of the replicative intermediate RNA. The RF co-sedimented at high speed from nonionic detergent extracts of virus-induced heavy membrane fractions along with the released individual inner membrane vesicles whose size of 75-100 nm as well as association with viral NS3 was revealed by immunoelectron microscopy. Viral RF remained nuclease-resistant even after ionic detergents solubilized the more refractory inner VP membrane. All of the viral RNA species became nuclease-sensitive following membrane disruption only upon prior trypsin treatment, suggesting that proteins coat the viral genomic RNA as well as RF within these membranous sites of flaviviral replication. These results collectively demonstrated that the newly formed viral genomic RNA associated with the VP are oriented outwards, while the RF is located inside the nonionic detergent-resistant vesicles.

Animals↗
PubMed1980

Catalysis of adenovirus DNA synthesis in vitro by DNA polymerase gamma.

DNA replication complexes were purified from adenovirus type 5 (Ad5)-infected HeLa cells. DNA synthesis by these complexes in vitro was extremely sensitive to the inhibitors dideoxythymidine triphosphate, N-ethyl maleimide and p-hydroxymercuribenzoate. The bound DNA polymerase was released from the complexes by limited digestion with micrococcal nuclease. This released polymerase preferred poly(rA):(dT)12-18 as template over activated calf thymus DNA. These results are compatible with the major polymerase in the replication complex being of the gamma class.

Adenoviridae↗
PubMed1999

Characterization of intracellular reverse transcription complexes of Moloney murine leukemia virus.

To examine the early events in the life cycle of Moloney murine leukemia virus (MoMLV), we analyzed the intracellular complexes mediating reverse transcription. Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that three distinct species of intracellular complexes are formed shortly after cell infection. Only one of these species is able to start and complete reverse transcription in the cell cytoplasm. This RTC is composed of at least the viral genome, capsid, integrase, and reverse transcriptase proteins. The RTC becomes permeable to micrococcal nuclease but not to antibodies. Shortly after initiation of reverse transcription, the viral strong stop DNA within the RTC is protected from nuclease digestion. The sedimentation velocity of the RTC decreases during reverse transcription. After entry into the nucleus, most capsid proteins are lost from the RTC and its sedimentation velocity decreases further.

Blotting, Western↗
PubMed1983

A simple method for the preparation of extracts from animal cells which catalyze efficient in vitro protein synthesis.

A rapid, simple procedure is described for the preparation of cell-free extracts of both uninfected and virus-infected pig kidney and baby hamster kidney cells which are very active in the in vitro translation of not only endogenous mRNA, but also of exogenous mRNA added to extracts that had been depleted of endogenous mRNA by treatment with micrococcal nuclease. This procedure appears to be adaptable with only minor variations to many eukaryotic cell lines and should greatly facilitate in vitro molecular studies of protein synthesis and gene regulation at the level of translation.

Animals↗