Inhibition of herpes simplex virus (HSV) thymidine kinase (TK) gene transcription (pHSV-106, pML-BPV-TK4) by DNA methylation is an indirect effect, which occurs with a latency period of -8 hr after microinjection of the DNA into TK-rat 2 and mouse LTK-cells. We have strong evidence that chromatin formation is critical for the transition of the injected DNA from methylation insensitivity to methylation sensitivity. Chromatin was reconstituted in vitro by using methylated and mock-methylated HSV TK DNA and purified chicken histone octamers. After microinjection, the methylated chromatin was always biologically inactive, as tested by autoradiography of the cells after incubation with [3H] Preparation of Histone Octamers and Chromatin Reconstitution. Chicken nuclei were isolated as described (16,17).Nuclei (3 x 1010) suspended in 0.25 M sucrose solution were diluted with an equal volume of 0.25 M HCl/0.2 M NaCl solution. After 30 min on ice, the suspension was centrifuged at 10,000 x g for 20 min and the supernatant was dialyzed twice against water and then freeze-dried. Core histones were isolated by treatment of the freeze-dried material with 5% perchloric acid (0.1 ml/mg) and centrifugation at 10,000 x g for 15 min. The pellet was reextracted in 2 ml of 0.25 M HCl and dialyzed twice against 0.25 M HCl and three times against water. The freeze-dried core histones (free of histones H1 and H5) can be stored at -20°C.To reconstitute histone octamer (17), the freeze-dried core histones were suspended in 8 M urea/1% (vol/vol) 2-mercaptoethanol to a final concentration of 4 mg/ml. After incubation for 2 hr at room temperature, the suspension was dialyzed against 2 M NaCl/1% 2-mercaptoethanol/10 mM Tris HCl, pH 7.4. The probe was then applied to a column (Sephadex 6B; 1 m x 2.5 cm), and eluted with 2 M NaCl/0.1 mM phenylmethylsulfonyl fluoride/10 mM Tris'HCl, pH 7.4, and the fractions containing the octamers were collected by measuring the absorbance at 280 nm. Final concentration was -0.1 mg/ml.For chromatin reconstitution, 10 ,g of DNA was mixed with 10 ,ug of reconstituted octamers in a final volume of 100 Al and dialyzed twice against 100 mM KCl/20 mM NaCl/0.25 mM EDTA/10 mM 2-mercaptoethanol/0.1 mM phenylmethylsulfonyl fluoride/10 mM Tris HCl, pH 7.4, for 48 hr. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Simian virus 40 (SV40) induces tumor (T)antigen formation, chromatin replication, and mitosis in primary mouse kidney cells arrested in Go phase of the mitotic cycle. The temporal and quantitative relation between these early virus-specific reactions led to the hypothesis that the early SV40 mRNA contains information necessary for T-antigen formation and induction of cellular DNA synthesis. To get direct experimental evidence for this hypothesis, the early strand of SV40 DNA was transcribed in vitro by Escherichia coli DNA-dependent RNA polymerase and the SV40-specific cRNA was transferred by microinjection into epitheloid cells of confluent primary mouse kidney cultures. T-antigen formation and stimulation of DNA synthesis were investigated in the recipient cells. The experimental results obtained agree with the hypothesis that T-antigen is a viruscoded protein and that the early virus-specific mRNA contains information necessary for stimulation of cellular DNA replication in the arrested cells.The infectious agent of simian virus 40 (SV40) is the virus DNA. The manner in which the virus genes are expressed is highly cell dependent. Cells of the native host (monkey cells) support early and late viral gene expression at a high efficiency (productive infection) while mouse kidney cells are nonpermissive for SV40 (abortive infection) (1, 2). However, the following sequence of events can be observed in both productive and abortive infected cells: (a) synthesis of early virus-specific mRNA, (b) formation of tumor (T)-antigen, (c) increase of cellular RNA synthesis, (d) induction of chromatin replication and mitosis (3).The temporal and quantitative relation between the synthesis of the early virus-specific RNA and the subsequent sequence of events led to the hypothesis that the early virusspecific RNA may contain information necessary for T-antigen formation and chromatin replication (3,4).In the present study, we have tested this hypothesis by a more direct experimental approach. For (Amersham Buchler,15 Ci/mmol). The nucleoside triphosphates were added after preincubation of SV40 DNA and RNA polymerase at room temperature for 10 min. After 60 min of incubation at 370, 10 ,g of electrophoretically purified DNase (Worthington) was added to the reaction mixture and further incubated for 30 min at 37°. Then 0.1 X SSC and 5% sodium dodecyl sulfate were added to a final concentration of 0.01 X SSC and 1% sodium dodecyl sulfate. The assay was extracted two times with 1 volume of phenol-hydroxyquinoline (80% phenol in H20 vol/vol and-l% hydroxyquinoline weight/volume) (12) and precipitated from the aqueous phase at -20°with 2 volumes of ethanol (95%) and 'Ao volumes of 1 M NaCl. To further reduce traces of any contaminating SV40 DNA, the cRNA preparation was subsequently subjected to Cs2SO4 equilibrium density gradient centrifugation (see Fig. 1) (6, 13). The average yield of cRNA was 80-100 Mug with a specific activity of 1.5 to 2 X 105 cpm/,g of cRNA. Self-an-366
We present evidence that mammalian cells have the ability to generate functional mRNA molecules by trans‐splicing. Rat cells, transformed by an early SV40 DNA fragment (Bst/Bam) synthesize a truncated T antigen (T1 antigen), although the cells do not have a direct sequence homology for the T1 antigen at the DNA level. The Bst/Bam DNA fragment encodes exclusively for the second SV40 T antigen exon (aa 83‐708) and contains the entire small t antigen intron. To synthesize the corresponding mRNA (T1 mRNA), the cells utilize a cryptic 5′ splice site within the second exon (codons for aa 131/132) as donor site and the upstream small t antigen 3′ splice site as the acceptor site. Since these sites are in an inverted order on the pre‐mRNA, two Bst/Bam transcripts are required to generate one T1 mRNA molecule. HeLa cell nuclear extracts also performed the trans‐splicing reaction in vitro.
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