Two regions of the adenovirus 2 genome specify families of late polysomal RNAs containing common sequences.

Abstract

Late cytoplasmic RNAs specified by two regions of the adenovirus 2 genome (39.3-51.8 and 70.7-83.4 The availability of specific DNA fragments has enabled the formation of transcriptional maps for adenovirus 2 RNAs synthesized both early and late in productive infection. By late times, after the onset of viral DNA replication, greater than 90% of the coding capacity of the virus is represented in cytoplasmic transcripts (1, 2). The viral mRNAs synthesized at typical late times, 18 hr after infection, are derived more than 99% from the r strand of the genome (3), the strand transcribed in the rightward direction on the conventional map (1). Transcriptional maps have been derived from two types of experiments: The number of nucleotides transcribed into mRNA has been determined by hybridization of nonradioactive RNAs with separated strands of radioactive DNA fragments (1, 2). mRNA species transcribed from various regions of the genome have been identified by hybridization analysis of radioactive RNAs separated by size (4-6).Mapping experiments with cytoplasmic viral RNAs synthesized late in infection revealed an unexpected size distribution for the RNAs transcribed from two separate regions of the viral genome (6). Each region specified a series of cytoplasmic RNAs whose collective sequence content exceeded the coding capacity of the region at least 2-fold. These two regions included the sequences contained in map positions 39.3-51.8 (Sma I-D DNA fragment) and 70.7-83.4 (EcoRI-F and -D DNA fragmepts). The region 39.3-51.8 appears to code forThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 625 several virion proteins, penton (85,000 daltons) pVII (20,000 daltons) and V (48,400 daltons) (7). The late proteins identified as products of the second region, 70.7-83.4, are pVIII (26,000 daltons) and a 100,000-dalton polypeptide.To better define the relationship between the series of RNAs transcribed from each of these regions, it was necessary to establish the presence of each of these RNAs in polyribosomes and to map in greater detail the sequences specifying each species. The mapping data presented here establish that, within each set of RNAs, sequences towards the 3' ends are shared, and the longer the molecule, the further it extends in the 5' direction. MATERIALS AND METHODSVirus Infection, Labeling of Cultures, and RNA Purification. Maintenance of KB cell suspension cultures and infections with adenovirus type 2 were performed as described previously (4,6). Cytoplasmic RNA was purified from cultures labeled with [3H]uridine (40 Ci/mmol; New England Nuclear at 50 ,uCi/ml. [3H]RNA labeled 12-14 hr after infection was utilized in mapping the cytoplasmic RNAs from the EcoRI-F,D region, and [3H]RNA labeled from 17-19 hr was used in analyzing the Sma I-D transcripts.Polyribosomes were isolated by sedimentation through a 7-47% sucrose gradient...

Analysis in Cos-1 cells of processing and polyadenylation signals by using derivatives of the herpes simplex virus type 1 thymidine kinase gene.

“…The major late transcription unit of adenovirus type 2 contains five sites for processing and polyadenylation, but only one is used each time the unit is transcribed (3,44,49,58,61,75). The relative abundance of different 3' coterminal mRNA families is determined in part by the proportion of processing and polyadenylation events occurring at each of the five sites.…”

Sequences upstream of AAUAAA influence poly(A) site selection in a complex transcription unit.

“…Temporal regulation of 3'-end formation is seen in the adenovirus major late transcription unit (MLTU), where five poly(A) sites define five colinear mRNA families, designated Li through L5 (42,47,53). Poly(A) site selection precedes splicing in the MLTU and is regulated during the course of infection such that usage of the Li site always differs from usage of the other sites.…”

“…Whether the dominant control mechanism is exerted at the level of poly(A) site selection (24,25), transcription termination (24), splice site-poly(A) site competition (56, 57), or some combination thereof is still being debated. Nevertheless, it is generally accepted that position and intrinsic strength of processing signals are critical for regulation.Temporal regulation of 3'-end formation is seen in the adenovirus major late transcription unit (MLTU), where five poly(A) sites define five colinear mRNA families, designated Li through L5 (42,47,53 …”

“…Temporal regulation of 3'-end formation is seen in the adenovirus major late transcription unit (MLTU), where five poly(A) sites define five colinear mRNA families, designated Li through L5 (42,47,53 …”

Historical Development of the Concept of the Gene

“…In certain cases, there are multiple alternative polyadenylation sites in the primary transcript. This was ®rst observed in adenoviruses (Klessig, 1977;McGrogan & Raskas, 1978;Nevins & Darnell, 1978;Wilson & Darnell, 1981;Ziff & Fraser, 1980). In cellular genes, many alternative polyadenylation sites have also been found (see Left et al, 1986 for review).…”