1. Temporal Patterns of Human Cytomegalovirus Transcription: Mapping the Viral RNAs Synthesized at Immediate Early, Early, and Late Times After Infection
2. H. Zhu and T. Shenk unpublished data.
3. Primers were designed to amplify all 208 predicted HCMV ORFs of 100 amino acids or more (4 23). The entire gene was amplified if it was equal to or less than 600 nucleotides in length. For genes greater than 600 nucleotides 300– to 400–base pair DNA fragments corresponding to the 3′ end of the gene were amplified. All DNA fragments were amplified by PCR with purified AD169 HCMV DNA as template. PCR products were gel purified and reamplified with the same primer pairs. DNA fragments were purified and then concentrations were determined. DNA fragments were denatured in 0.8 M NaOH and 20 mM EDTA (pH 8.2) and 5 ng of each gene was spotted onto Nytran+ nitrocellulose membranes with a 386-pin multiblot replicator (VP-Scientific San Diego CA). DNA was ultraviolet–cross-linked to the membranes. PCR products were cloned into the pGEMT-Easy vector (Promega Madison WI) so subsequent amplifications could be performed with a universal primer set.
4. Chee M. S., et al., Curr. Top. Microbiol. Immunol. 154, 125 (1990).
5. DNase I–treated RNA was isolated from virus particles that were partially purified by centrifugation through a 20% sorbitol cushion (11) and treated with RNase One (Promega Madison WI) to degrade RNA bound externally to the particles. Virion RNA was reverse transcribed in the presence of [α- 32 P]deoxycytidine triphosphate to generate a radioactively labeled cDNA.