Isolation of DNA Strand-Specific Early Messenger RNA Species in Cells Infected by Human Adenovirus 2

Abstract

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...

In vitro synthesis of adenovirus type 5 T antigens. I. Translation of early region 1-specific rna from lytically infected cells

“…Cytoplasmic RNA was isolated from KB cells lytically infected with Ad5 (6 h after infection) essentially according to Flint et al (6). Viral RNA was selected by hybridizing 1 mg of cytoplasmic early RNA to 20 pg ofAd5 DNA immobilized on nitrocellulose filters (8), and RNA was eluted essentially according to Buttner et al (3). The RNAs were translated in a wheat germ cell-free system.…”

Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation.

“…DNA, either in liquid or bound to immobilized supports, has been used to hybridize and thereby sequester specific mRNAs from the plethora of cellular RNA species. These methods have included isolation of RNA-DNA hybrids by: selective binding to hydroxylapatite (1); selective exclusion through agarose (2) or Sepharose 4B (3); the use of DNA covalently bound to cellulose (4,5) or Sepharose (6); DNA bound directly to nitrocellulose (7)(8)(9); DNA enzymatically synthesized and covalently bound to oligo(dT)-cellulose (10).…”

Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution

“…(9) was applied to selections with 8.7-10.7 DNA. After hybridization the membranes were washed as described and bound RNA was eluted at 850.…”

“…Precipitated RNA was spun down at 25,000 rpm, the pellet dried under nitrogen, and resuspended in [5][6][7][8][9][10] (26) with an acrylamide:bisacrylamide ratio of 30:0.174. Slab gels were 1.5 mm thick, with a 5% stacking gel 1.5 cm in length, and an 8 cm resolution gel of 17.5%.…”

“…In light of recent findings that demonstrate the presence of multiple inserts within the coding sequences of a gene (1)(2)(3)(4) and show that a specific gene sequence may express different products by synthesizing overlapping mRNAs (5)(6)(7)(8), it is often necessary to identify functional genes by purifying the mRNA coding for the gene product. Several methods have been described for the preparative isolation of specific RNA molecules by hybridization to DNA immobilized on a matrix (9)(10)(11)(12), or by hybridization to DNA in liquid and purification of the DNA-RNA hybrid (13). The nitrocellulose membrane technique has several advantages: DNA can be bound to membranes by rapid and efficient methods (14) and the DNA membranes, which may contain large amounts of valuable DNA, are reusable.…”