Adenovirus 2 early messenger RNA-genome mapping of 5'-terminal RNase T1 oligonucleotides and heterogeneity of 5'-termini.

The human adenovirus (Ad) type 2 genome (Mr = 23 X 10‘) is expressed in permissive human cells in two major stages, early and late. Early genes are organized in four major regions (El to E4) located in noncontiguous regions of the viral genome. To define the number of different 5’-termini, we have isolated, characterized, and mapped in E l to E4 the 5”terminal oligonucleotides produced by RNase T1 digestion of Ad2 early mRNA. Poly(A+) polyribosomal RNA was isolated from Ad2infected cells, the 5’-terminal m’Gppp was removed, and the 5’-OH of the penultimate 2’-O-methylated nucleoside was labeled with [y3’P]ATP using polynucleotide kinase. Total Ad2 5’-32P-labeled early RNA was then isolated by two cycles of hybridization to Ad2 DNA. E l to E4 specific RNA was selected by further hybridization to Eco RI DNA restriction fragments. Purified 5’-32P-labeled Ad2 mRNA preparations were digested with RNase T1, the resulting oligonucleotides resolved by two-dimensional paper electrophoresishomochromatography, and the 5’4erminal methylated nucleotides characterized. We mapped 13 5’-termini on Ad2 DNA, three in Eco RI-A (El), one in Eco RI-€3 (E2), three in Eco RI-D (E3), and six in Eco RI-C (E4). Since only one major promotor has been localized in E3 and E4, E3 and E4 mRNA have heterogeneous 5’-terminal sequences. The heterogeneous 5’4ermini in E3 and E4 may be due to “stuttering” by RNA polymerase 11, different promotor-initiation sites, or endonucleolytic processing. Although most Ad2 mRNAs initiate in purines (A” or G”), three either initiate in a pyrimidine (Urn), or are processed to yield 5”penultimate u”. Of interest, the Ad2 mRNAs with penultimate u” were observed only in E4. At least 11 different 5’-terminal T1 fragments were detected in uninfected KB cell mRNA; only two of these could be common to Ad2 mRNA.