Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids

Abstract

We have developed a simple and sensitive method for detecting, sizing and mapping RNA transcripts from viral or cloned DNAs. This technique has been used to examine the cytoplasmic transcripts produced during the early phase of adenovirus 2 (Ad2) infection of HeLa cells. Unlabeled total cytoplasmic or oligo (dT)-selected cytoplasmic RNA is hybridized to restriction fragments of 32P-labeled viral DNA in 80% formamide under conditions above the Tm of the DNA duplex, but below the Tm of the RNA-DNA hybrid duplex ( Casey and Davidson, 1977). DNA complements precisely the length of the hybridized RNA are generated by treating with single-strand-specific S1 endonuclease under conditions which do not introduce strand breaks into hybrid duplex. The sizes of the S1-resistant single-stranded DNAs are then determined by alkaline agarose gel electrophoresis ( McDonnell, Simon and Studier, 1977). A restriction fragment which terminates within a region coding for an mRNA yields a band equal in size to the portion of the mRNA transcribed from that restriction fragment. This allows unique mapping of coding regions relative to restriction endonuclease cleavage sites. All early Ad2 transcripts are clustered in four regions of the viral DNA. At least five early stable cytoplasmic colinear transcripts are transcribed to the right from the left end of the viral genome, the region of the genome coding functions necessary for transformation of mammalian cells. Transcripts of 650, 350 and 1750 nucleotides map from 1.7 ± 0.5 to 3.6 ± 0.5, 3.6 ± 0.2 to 4.6 ± 0.4, and 4.7 ± 0.3 to 9.5 ± 0.5 units, respectively, and there are two 450 nucleotide transcripts tentatively mapped in the region of 3.0 and 11.0 units. Two overlapping transcripts of 1600 and 1700 nucleotides having the same 3′ terminus and 5′ termini displaced by 100 nucleotides are transcribed to the left from 66.2 ± 0.3 to 61.6 ± 0.2 and 66.5 ± 0.3 to 61.6 ± 0.2 units, respectively. Seven other distinct early stable cytoplasmic transcripts have also been positioned on the genome.