Hybridization to the separated light (L) and heavy (H) strands of adenovirus 2 DNA in 50% formamide at 37°was used to isolate undegraded virus-specific RNA molecules from the polyribosomes of cycloheximidetreated human KB cells early after infection with adenovirus 2. About 20% of polyribosomal RNA labeled with [3Hluridine from 4 to 7 hr after infection was virus-specific. Twice as much labeled RNA was homologous to the L strand as to the H strand. Polyacrylamide gel electrophoresis of RNA selected with unfractionated adenovirus DNA resolved a major component of virus-specific RNA in the 19-20 S region of the gel and smaller amounts of viral RNA in two heterogeneous fractions migrating at 15-18 S and 21-26 S. Selection with individual DNA strands showed that the 19-20 S main size class of early mRNA consists of two homogeneous RNA species with slightly different mobilities, the transcripts from the L and H strand having molecular weights of 7.4 X 105 and 7.7 X 105, respectively. The 15-18 S RNA hybridized with the L strand and the 21-26 S RNA with the H strand.Only one of the two complementary DNA strands in any region of a duplex DNA molecule can be transcribed to a functional messenger RNA for protein synthesis. With some DNA viruses transcription occurs exclusively from one strand of the viral genome (1), while with others regions from both strands are transcribed (2, 3). The human adenoviruses, especially the well-studied adenovirus type 2, form excellent models for study of the regulation of transcription in mammalian cells. Of special interest is the transcription of early adenovirus 2 genes that occurs in the absence of protein synthesis and thus probably reflects the transcril)-tion controls of the host cell (4). Early after infection only a few viral genes are transcribed to stable viral mRNA species (5), and these include those adenovirus genes expressed in transformed cells (6). It would be of interest, therefore, to be able to isolate individual adenovirus mRNA molecules in an undegraded form for studies on structure, sequence, and translation. The use of the separated strands of adenovirus DNA facilitates resolution of individual viral mRNA species. Landgraf-Leurs and Green (7) developed a procedure for the preparative separation of the adenovirus tylpe 2 DNA strands, and showed that both strands were transcribed early as well as late after infection (8). Viral RNA isolated by hybridization to DNA by the usual annealing conditions is thermally degraded and cannot be used for further characterization.Abbreviations: L and H strand, light and heavy strands as defined by the relative buoyant density of the DNA strand. poly(U,G) complex; EDTA, ethylenediaminetetraacetate. 2951Although undegraded adenovirus-specific RNA can be isolated on a preparative scale on the basis of its content of poly-(adenylic acid) (9), this approach is useful only for the isolation of late viral RNA species which contain relatively little cell mRNA. Molecular hybridization to viral DNA is required to separate early vir...
The early genes of the human adenotirus type 2 (Ad 2) expressed prior to the onset of DNA replication at 6 h post-iifection, are involved in regulatory functions necessary for the virus growth cycle and in the oncogenic transformation of mammalian cells. After initial transcription in the nucleus by host cell mechanisms, the early viral RNA species are modified by polyadenylation on the 3'-end, addition of a S'methyl cap (m'Gppp) and excision of intervening sequences (sp~c~g) to yield mature polysomal mRNA species. The physical map of the early Ad 2 genes reveals 4 separate gene regions, El -E4, located at the map coordinates 1.5-I 1 (El) and 76-86 (E3) on the r-strand, and 68-62 (E2) and 99-91.5 (E4) on the I-strand (r and 1 indicate rightward and leftward transcription of the Ad 2 genome, presented in 100 map units). Each early gene block codes for a family of spliced partially overlapping mRNA species, usually with common 3'end 5'-termini [l-6]. (Two additional sets of early viral RNA sequences may be present in low copy number, complementary to the map regions 11-14.5 and 20-23.5 on the f-strand 171.) The gene product of the region E2 is a DNA-bin~mg protein (M, 73 000) involved in Ad 2 DNA replication [8,9] and perhaps late gene expression [IO]. The characterization of the mRNA of this DNA-binding protein (DBP) is therefore of particular interest. We had calculated the coordinates of the main body of this mRNA by determining the % of radioactivity of DBP mRNA which hybridized to particular subfrag ments of the EcoRI B fragment (map position 58.5-70.7) ill ]. Our findings are in good agreement with the data obtained by an Sr nuclease technique [ 121 and by electron microscopy [6]. We showed that the Elsevier/North-Holland Biomedical Press 3'-terminus of the DBP mRNA lies within the restriction fragment BgiII J (60. 2-63.6) at the map position 61 .I C 0.4. Here, we prepared A&I and N&r11 subfragments of BglII J, and by hybridization to small poly(A)-containing fragments of the DBP mRNA isolated an -300 basepair subfragment which is homologous to the 3'-terminal region of this mRNA. Analysis of the primary structure of this subfragment identified the DNA sequence which codes for the 3'-terminus of the DBP mRNA. Materials and methods Restriction endonucleasesRestriction endonucleases EcoRt, BglII, HpaII, AU, KpnI, HaeIII, BaZI and ma1 [ 131 were isolated by standard biochemical procedures including ammonium sulfate fractionation, phosphocellulose, DEAEcellulose and hydroxylapatite chromatography, gel filtration, heparin-agarose and aminoalkyl-agarose affinity chromato~aphy.AvaI and HphI were purchased from New England BioLabs. DNA restriction fragmentsThe preparation of Ad 2 DNA,cleavage with restriction endonucleases and the isolation of the viral DNA fragments from 1.4% agarose gels has been described [3]. Purification of small restriction fragments from polyacrylamide gels was done as in [ 143.2.3.~xI75RFIDMA RF I DNA of #xl 74~723 was isolated as suggested by W. Riiger, Boch~.~aeIII fra~ents of@xl74am3[ ...
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