ever, cannot multiply in baby hamster kidney. (BHK-21) cells (3). The block in viral replica- tion is early; i.e., newly synthesized viral DNA cannot bedetected in ...
Vol. 22, No. 3 Printed in U.S.A.
JOURNAL OF VIROLOGY, June 1977, p. 585-590 Copyright © 1977 American Society for Microbiology
Transcription of the Genome of Adenovirus Type 12 IV. Maps of Stable Late RNA from Productively Infected Human Cells KARL-HEINZ SCHEIDTMANN AND WALTER DOERFLER* Institute of Genetics, University of Cologne, Cologne, Germany Received for publication 19 November 1976
From human KB cells productively infected with adenovirus type 12, mRNA and stable nuclear RNA were isolated late (42 h) after infection. Using restriction endonuclease fragments of adenovirus type 12 DNA, mRNA and stable nuclear RNA sequences were mapped on the viral genome. Late after infection, preferentially the r (= rightward) strand is transcribed into stable nuclear RNA, whereas the 1 (= leftward) strand is expressed only to a minor extent. Adenovirus type 12-specific mRNA originates from the following sections on the viral genome: 0 to 0.11, 0.18 to 0.20, 0.27 to 0.49, 0.56 to 0.63, 0.68 to 0.84, and 0.89 to 0.92 fractional length units on the r strand and 0.11 to 0.16, 0.22 to 0.27, 0.50 to 0.54, 0.62 to 0.66, 0.855 to 0.865, and 0.93 to 1.0 fractional length units on the 1 strand. Self-complementary viral RNA isolated at 8 h postinfection is complementary to about 20% of each strand of the viral genome, and self-complementary viral RNA isolated at 42 h postinfection anneals to 70 to 80% of each strand of the viral genome.
Adenovirus type 12 (Adl2) productively infects human KB cells. In this permissive system, viral DNA replication starts between 12 and 14 h postinfection (p.i.) (14). Adl2, however, cannot multiply in baby hamster kidney (BHK-21) cells (3). The block in viral replication is early; i.e., newly synthesized viral DNA cannot be detected in the nonpermissive system with the most sensitive methods (4, 6). Early genes of the Adl2 genome are expressed in BHK-21 cells (10, 13). By using restriction endonuclease fragments of Adl2 DNA, mRNA and stable nuclear RNA from Adl2-infected BHK-21 cells and from two lines of Adl2-transformed hamster cells have been mapped on the viral genome (10). It has been shown that these RNA populations originate from the same regions of the Adl2 genome as the mRNA and stable nuclear RNA found early in productively infected cells (10). To complement these earlier studies, we have now determined the map positions of the Adl2specific RNA synthesized late in productive infection. Work on the mapping of Adl2-specific RNA from productively infected cells has also been carried out by Smiley and Mak (16). These authors reported that, late during productive infection, cytoplasmic RNA hybridized to 65% of the L (= light) strand = r strand and 15% of the H (= heavy) strand = 1 strand. Late nuclear RNA was complementary to about 90% of the r strand and 36% of the 1 strand. Late after infection, Smiley and Mak found double-stranded RNA that hybridized to >30% of both Adl2
strands. Early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. The availability of transcriptional maps of Adl2 in different systems allows one to compare the location of viral genes expressed early and late in infection in the Ad2 and Adl2 genomes. Precise transcriptional maps of the Ad2 genome have been designed based on work performed in several laboratories (12, 15). In general, the orientation and location of functionally equivalent genes, i.e., genes that are expressed early or late, appear to be congruent in the two adenovirus genomes, although Ad2 and Adl2 belong to different oncogenic classes of adenoviruses. In the present report the maps of the Adl2specific mRNA and stable nuclear RNA synthesized late in productive infection were determined by saturation hybridization experiments, using specific restriction endonuclease fragments of Adl2 DNA. We have also started to investigate to what extent both strands of the viral genome are transcribed symmetrically into stable nuclear RNA. MATERIALS AND METHODS Most of the techniques used in this report have been described previously (3, 9, 10, 14) and will not be repeated here. Cell lines and virus. Human KB cells (5) were grown in monolayer or suspension cultures. Human Adl2 was propagated in suspension cultures of KB cells and purified as described earlier (3, 9, 10). 585
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Inoculation of KB cells with Adl2. KB cells growing in suspension culture were infected with CsClpurified Adl2 at multiplicities of 100 PFU/cell (9). At various times after infection, the polysomes or the nuclei were isolated as described elsewhere (9, 10). Isolation of mRNA and stable nuclear RNA. These methods were also outlined earlier (10). mRNA containing polyadenylic acid [poly(A)] sequences was selected by affinity chromatography on polyuridylic acid [poly(U)]-Sepharose. Nuclear RNA was isolated by the method of Holmes and Bonner (7) with minor modifications. Infected KB cells growing on monolayers were pulse-labeled with [53H]uridine (100 ,uCi/ml, 23 to 26 Ci/mmol) for 10 min. After the pulse, the cells were lysed in 0.5% Nonidet P-40 in isotonic buffer, and the nuclei were pelleted by low-speed centrifugation. The nuclei were resuspended in 0.35 M NaCl-0.001 M EDTA0.01 M Tris-hydrochloride (pH 8.0) and lysed at room temperature by the addition of 1% each of sodium dodecyl sulfate (SDS) and Sarkosyl. After three cycles of phenol extraction, the nucleic acids were precipitated with 2 volumes of ethanol at -20°C for 12 h. The precipitate was treated with DNase (50 ,ug/ml) at 37°C for 5 to 15 min as described earlier (10). Subsequently, the reaction mixture was extracted twice with phenol, and the RNA was precipitated with ethanol. Analysis of the stable nuclear RNA by velocity sedimentation in sucrose density gradients containing 50% formamide. To analyze the size of the nuclear RNA, the precipitated material was dissolved in 90% formamide in 0.1 M LiCl-0.005 M Tris-hydrochloride (pH 7.5)-0.005 M EDTA-0.2% SDS (buffer A) and heat denatured at 37°C for 5 min (1). Subsequently, the RNA was quickly cooled to 0°C, diluted with 1 volume of buffer A, and sedimented in a 5 to 20% sucrose density gradient in 50% formamide in buffer A in an SW56 rotor of a Spinco L2-65B ultracentrifuge at 45,000 rpm and 4°C for 5 h (1). The fractions collected were assayed for Adl2-specific RNA by DNA-RNA filter hybridization as described previously (9, 10). Principle of RNA mapping experiments. Experimental details have recently been described by Ortin et al. (10). Briefly, the complementary strands of each of the EcoRI or BamHI restriction endonuclease fragments of 3H-labeled Adl2 DNA were prepared by equilibrium centrifugation in poly(U,G)-CsCl density gradients. The two complements of Adl2 DNA are designated the H (heavy) and L (light) strands, as they bind relatively greater and lesser amounts of poly(U,G), respectively. The map of Adl2 DNA, its polarity, and the position of the EcoRI and BamHI restriction endonuclease fragments have been determined previously (10) and are shown in Fig. 1. Since the heavy complements of some of the restriction endonuclease fragments from the left end of the Adl2 genome are part of the intact L strand and vice versa (10), the designations r (= rightward) strand for the L strand and 1 (= leftward) strand for the H strand will be used throughout the manuscript (Fig. 1). Each of the 3Hlabeled complements was annealed in a liquid-phase
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FIG. 1. Schematic representation of the Adl2 DNA molecule. The polarities of the H = heavy (leftward transcription) and L = light (rightward transcription) strands relative to the restriction maps of Adl2 DNA for the EcoRI and BamHI endonucleases are indicated. The H strand is designated the 1 strand, and the L strand is designated the r strand. The designations H (heavy) and L (light) refer to the strands binding relatively larger and smaller amounts of poly(U,G) in CsCl density gradients. There is a "reversal in heaviness" on the left terminus of the Adl2 DNA molecule as described earlier (10). The scale on the bottom gives fractional length units of the Adl2 DNA molecule.
hybridization system (10) with unlabeled mRNA or stable nuclear RNA isolated from Adl2-infected KB cells late after infection. The extent of complementarity between the RNA fractions tested and each of the complements of specific Adl2 DNA fragments was measured by incubating the hybridization product with the single-strand-specific nuclease S1 at the end of the hybridization reaction. The undigested DNA-RNA hybrid was precipitated with trichloroacetic acid and counted on GF/C filters. Saturating amounts of RNA in the annealing reaction were assessed experimentally and calculated by the method of Lucas and Ginsberg (8). The percentage of 3H-labeled fragment complement converted into an S1-resistant DNA-RNA hybrid has been calculated and plotted directly in the maps shown in Fig. 3. In control experiments,
