Species Identification and Genome Mapping of Cytoplasmic Adenovirus Type 2 RNAs Synthesized Late in Infection

Abstract

Adenovirus type 2 cytoplasmic RNAs synthesized late in productive infection were resolved by electrophoresis on formamide gels. Regions of the adenovirus 2 genome specifying RNAs of distinct size were determined by hybridization to specific DNA fragments generated by cleavage with endo R EcoRI and endo R * SmaI. From these studies 13 distinct viral RNA species were identified. A 26S to 28S size class and a 21S to 23S size class were each found to consist of four distinct RNA species. Three RNA species were identified in a 16S to 18S size class, and a fourth size class, 11S to 13S, was resolved into two components. The SmaI-D region (0.38 to 0.51 on the unit genome) and the EcoRI-F, D region (0.70 to 0.83) of the genome were found to code for multiple transcripts. Three RNAs (28S, 22S, and 18S) are specified by SmaI-D, and four components, 28S, 22S, 18S, and 16S, are encoded by EcoRI-F,D. The RNA represented by each set of multiple transcripts exceeds the coding capacity ofthe respective region, and the species within each set of RNAs appear to contain common sequences. The relationship between the cytoplasmic RNA species synthesized at late times and early cytoplasmic RNAs was determined by hybridization-inhibition experi-240

Expression of adenovirus-2 early region 4: assignment of the early region 4 polypeptides to their respective mRNAs, using in vitro translation

“…Virus infections and RNA isolation. Infections were performed as previously described (27). When cycloheximide (Sigma Chemical Co.) was used to inhibit protein synthesis, 25 ,ug of cycloheximide per ml was added 1 h after infection, and the cells were harvested at 7 h. In some experiments, cytosine arabinoside (araC; 20 F,g/ml; Sigma) was added 1 h after infection, and the cells were harvested at 5 h. In all experiments, RNA was labeled with [5,6_3H]uridine (2.5 to 10 ,Ci/ ml; 50 Ci/mmol; New England Nuclear Corp.) added 2 h before the cells were harvested.…”

“…When cycloheximide (Sigma Chemical Co.) was used to inhibit protein synthesis, 25 ,ug of cycloheximide per ml was added 1 h after infection, and the cells were harvested at 7 h. In some experiments, cytosine arabinoside (araC; 20 F,g/ml; Sigma) was added 1 h after infection, and the cells were harvested at 5 h. In all experiments, RNA was labeled with [5,6_3H]uridine (2.5 to 10 ,Ci/ ml; 50 Ci/mmol; New England Nuclear Corp.) added 2 h before the cells were harvested. The procedures used for isolating poly(A)-containing cytoplasmic RNA have been described elsewhere (27). When RNA was to be isolated from polysomes, infected cells were first treated with cycloheximide essentially as described previously (18).…”

Nucleosome-like structural subunits of intranuclear parental adenovirus type 2 DNA

“…Gel electrophoresis of DNA and DNP. Nuclear DNA purified after micrococcal nuclease digestion and adenovirus 2 DNA digested with endo R.SmaI (26) were resolved on either 1.4% agarose slab gels (0.3 by 18 by 16 cm) or 2.0% agarose tube gels (0.7 by 12 cm) as described previously (10). Electrophoresis was performed overnight at 50 V at room temperature for slab gels and 8 h at 1.5 mA per tube for cylindrical gels.…”

Group and type antigens of Capnocytophaga

“…The development of a single precipitation band was observed, and the appropriate strip of gel containing the antigen from wells 2 through 8 was collected. The antigen was extracted from the gel by three successive freeze-thaw cycles, followed by forcing the gel through an unfiltered syringe and finally through a glass wool-filtered syringe (10). The clear filtrate from eight such preparative IEP runs was pooled, dialyzed in the cold against distilled water, and then lyophilized.…”