PREPARATIVE ISOLATION AND MAPPING OF ADENOVIRUS-2 EARLY MESSENGER-RNA SPECIES

Five major early messenger RNA species with apparent MW (estimated by formamide gel electrophoresis) of 1.4 .times. 106 (24 S), 9.4 .times. 105 (20.5 S), 8.4 .times. 105 (20 S), 8.4 .times. 105 (20 S) and 4.2 .times. 105 (14 S) were isolated from adenovirus 2-infected [human oval carcinoma] KB cells. RNA was extracted from the polysomes of cells labeled from 2.5-5.5 h postinfection in the presence of cycloheximide to enhance the synthesis of early viral mRNA, and to prevent the synthesis of late mRNA. DNA was hybridized to and eluted from the DNA restriction fragments generated by [restriction] endonucleases EcoRI, SmaI [from Serratia marcescens], HindIII and HpaI. The conventional map of the linear adenovirus 2 DNA genome of 23 .times. 106 MW is oriented from left to right, map positions 0-1.0, with the r strand (rightward transcription) on top and the l strand [leftward transcription] on the bottom. The following mRNA species were selected: (1) both 24 S and 14 S RNA species from 5 partially overlapping subfragments of EcoRI-A, representing its left end segment (.apprx. 24% of the adenovirus 2 genome), SmaI-J (map position 0-0.030), SmaI-E/HindIII-G (0.030-0.075), SmaI-E/HindIII-C (0.075-0.11), HpaI-E (0-0.041) and HpaI-C (0.041-0.24); early RNA did not hybridize to SmaI-F (0.12-0.18), indicating that the right terminus of this early gene block terminates to the left of map position 0.12; this region contains genes for transformation; (2) a 20 S RNA, transcribed from the EcoRI-B fragment (map position 0.59-0.72); (3) a 20.5 S RNA from 2 subfragments of EcoRI-C (0.90-1.00), EcoRI-C/SmaI-C (0.90-0.92) and SmaI-G (0.92-0.98) (the terminal right SmaI-K (0.98-1.0) subfragment did not code for early mRNA); (4) a 20 S RNA, transcribed from EcoRI-D (0.76-0.84) and from a very small fraction of EcoRI-E (0.84-0.90). Several small viral RNA species were also detected whose significance was not established. The viral mRNA species transcribed from the EcoRI fragments A, D and E were specific for the adenovirus 2 r strand and the mRNA species of the EcoRI fragments B and C for the adenovirus 2 l strand. Rehybridization experiments indicated that the EcoRI fragment specific RNAs are unique with the exception of the 20 S RNA species most likely are encoded in the left 11% of the genome, 1 specific to the HpaI-E fragment and 1 to the HpaI-C fragment; the 24 S and 14 S species from EcoRI-A subfragments share sequences, suggesting that the small RNA may be derived from the large 1 by endonucleolytic cleavage.