Synchronized chicken embryo fibroblasts, prepared by addition of serum to stationary cells arrested in G., were exposed to the Prague strain of Rous sarcoma virus. At different times during the cell cycle, high molecular weight DNA was prepared from infected cells and examined for the presence of newly integrated viral DNA sequences. The results demonstrate that newly integrated viral sequences were first detected during Sphase DNA synthesis 9 hr after infection. The presence of colchicine prevented cellular division and delayed the appearance of progeny virus but it did not affect the appearance of viral specific DNA in the high molecular weight fraction of cellular DNA. Our results indicate that provirus integration, occuring during S-phase DNA synthesis, does not require cell division. Previous experiments have demonstrated that Rous sarcoma virus infection of chicken embryo fibroblasts requires cell division to initiate viral RNA synthesis and the production of progeny virus. The findings presented in this report support the hypothesis that division of the infected cell is required for an event that controls viral expression at the level of the integrated provirus.The replication of Rous sarcoma virus (RSV) proceeds through the formation and integration of a DNA copy of the viral genome, the provirus (1). Initiation oftranscription ofthe provirus and the production of infectious virus require an initial division of the newly infected cell (2, 3). However, when cells are arrested in Go after the production of progeny RSV has already begun, both the transcription of viral RNA and the production of virus continue in the absence of further cell division (3, 4). The requirement for cell division, therefore, is not continuous and appears to be an event necessary for activation of the provirus (5).Analysis of the early events in provirus formation has demonstrated that viral DNA synthesis is initiated after RSV infection of stationary cells (6). Examination of the viral DNA synthesized in these cells has revealed that it is incomplete and noninfectious (7). Complete synthesis of the provirus and its integration into cellular DNA has been shown to require an undefined host cell function(s) (8). To determine whether this host function(s) is related to the event(s) required for activation of the provirus, we analyzed the integration of the provirus in synchronized cells. The results presented below indicate that the complete provirus can be synthesized and integrated during S-phase DNA synthesis. Although the presence of colchicine inhibits the production of progeny virus, it has no effect on either the synthesis or integration of the provirus. It is likely, therefore, that the host function(s) required for the synthesis and integration of viral DNA is distinct from that required to activate the provirus. It seems that activation of the provirus depends on an event(s) that follows mitosis because colchicinetreated cells arrested in metaphase contain an inactive provirus.EXPERIMENTAL PROCEDURES Cells and Virus...
Over the past 10 years, polyvalent DNA–gold nanoparticle (DNA–GNP) conjugate has been demonstrated as an efficient, universal nanocarrier for drug and gene delivery with high uptake by over 50 different types of primary and cancer cell lines. A barrier limiting its in vivo effectiveness is limited resistance to nuclease degradation and nonspecific interaction with blood serum contents. Herein we show that terminal PEGylation of the complementary DNA strand hybridized to a polyvalent DNA–GNP conjugate can eliminate nonspecific adsorption of serum proteins and greatly increases its resistance against DNase I-based degradation. The PEGylated DNA–GNP conjugate still retains a high cell uptake property, making it an attractive intracellular delivery nanocarrier for DNA binding reagents. We show that it can be used for successful intracellular delivery of doxorubicin, a widely used clinical cancer chemotherapeutic drug. Moreover, it can be used for efficient delivery of some cell-membrane-impermeable reagents such as propidium iodide (a DNA intercalating fluorescent dye currently limited to the use of staining dead cells only) and a diruthenium complex (a DNA groove binder), for successful staining of live cells.
The DNA binding and cellular localization properties of a new luminescent heterobimetallic Ir(III) Ru(II) tetrapyridophenazine complex are reported. Surprisingly, in standard cell media, in which its tetracationic, isostructural Ru(II) Ru(II) analogue is localized in the nucleus, the new tricationic complex is poorly taken up by live cells and demonstrates no nuclear staining. Consequent cell-free studies reveal that the Ir(III) Ru(II) complex binds bovine serum albumin, BSA, in Sudlow's Site I with a similar increase in emission and binding affinity to that observed with DNA. Contrastingly, in serum-free conditions the complex is rapidly internalized by live cells, where it localizes in cell nuclei and functions as a DNA imaging agent. The absence of serum proteins also greatly alters the cytotoxicity of the complex, where high levels of oncosis/necrosis are observed due to this enhanced uptake. This suggests that simply increasing the lipophilicity of a DNA imaging probe to enhance cellular uptake can be counterproductive as, due to increased binding to serum albumin protein, this strategy can actually disrupt nuclear targeting.
Purified MuMTV spot 1 RNA was subjected to complete hydrolysis with either RNase T1 or pancreatic RNase, and the resulting oligonucleotides were separated by two-dimensional electrophoresis (Fig. 3). Each oligonucleotide was further characterized by redigestion with RNases of different specificity.
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