Regulation of the appearance of cytoplasmic RNAs from region 1 of the adenovirus 2 genome

The time-course of appearance of cytoplasmic RNA species coded by the region 9 to 10.7 on the adenovirus 2 genome (early region 1) has been examined using specific DNA probes. Hybridization with restriction endonuclease fragments of viral DNA resolved five cytoplasmic RNAs. A 22 S RNA contains sequences to the right of map position 4.4. One 13 S RNA (13 S_L) maps to the left of this site, while an RNA of similar size (13 S_f) is transcribed from two separate regions to the right of 4.4. Each 13 S RNA shares 3′ sequences with a distinct 9 S RNA. Sequences in 13S_f RNA are also present in the 22 S species. These mapping studies have allowed the identification of probes that can be used to assay each RNA after fractionation by size.During productive infection, each RNA species has a characteristic rate of appearance in the cytoplasm; 22 S and 13 S_L RNA accumulate at a constant rate throughout infection. Initially 13 S_f RNA appears at a rate substantially lower than the 22 S and 13 S_L, RNAs. At late times accumulation of 13 S_f RNA is stimulated more than 50-fold, until its appearance exceeds that of the other “early” RNAs. The 9 S RNAs are detected only after viral DNA replication begins. The rate of labeling of each approaches that of its colinear 13 S RNA. The results demonstrate a complex regulatory pattern for region 1 RNAs. Regulatory events must account for: (1) continued synthesis of early RNAs at late times; (2) increased accumulation of 13 S_f RNA; (3) the turn on of 9 S synthesis; and (4) balanced rates of appearance of the colinear 13 S and 9 S RNAs at late times.     

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.     

Identification of early adenovirus type 2 RNA species transcribed from the left-hand end of the genome.

Unique fragments of adenovirus type 2 DNA generated by cleavage with endonuclease R-Eco RI or endonuclease R-Hsu I (Hin dIII) were used to map cytoplasmic viral RNAs transcribed early in productive infection. Radioactive early viral RNA was first fractionated by polyacrylamide gel electrophoresis. Eluted viral RNAs were then tested for hybrid formation with DNA fragments. The Eco RI DNA fragment (Eco RI-A) which contains the left-hand 58% of the genome hybridized 13S and 11S RNAs. More detailed mapping of these RNAs was achieved by hybridization to the seven Hsu I fragments of Eco RI-A. The early RNA annealed only to Hsu I-G and C, two fragments which comprise the extreme left-hand 17% of the genome. Viral RNA migrating as 13S and 11S annealed to Hsu I-G, and 13S RNA annealed to Hsu I-C. A 13S RNA is transcribed from Eco RI-A late in infection (18 h). Hybridization-inhibition studies with Eco RI-A DNA, early cytoplasmic RNA, and 3H-labeled 13S late RNA demonstrated that this RNA synthesized at late times is an early RNA species which continues to be synthesized in large amounts at 18 h. This 13S RNA synthesized at 18 h hybridized to Hsu I-C but not to Hsu I-G DNA. These results establish that the 13S RNAs transcribed from Hsu I-G and C at early times must be different species.