The In Vitro Replication of Adenovirus DNA
Adenovirus (Ad) DNA is a linear double-stranded molecule of approximately 36000 bp with a covalently bound 55-kilodalton (K) terminal protein (TP) linked to the 5′ terminus of each strand (Robinson et al. 1973; Rekosh et al. 1977; Carusi 1977). Ad DNA has inverted terminal repeats of about 100 bp with the exact length depending on the serotype (Steenbergh et al. 1977; Arrand and Roberts 1979; Shinagawa and Padmanabhan 1979). A highly conserved sequence extending from nucleotides 9 to 22 from either end of the molecule may serve as a recognition site for proteins involved in DNA replication (Shinagawa and Padmanabhan 1980; Tolun et al. 1979). In addition, the 5′-terminal dCMP residue is conserved in all serotypes sequenced. The TP is attached via a phosphodiester bond between a β-hydroxyl group of a serine residue in the protein and the 5′-phosphate end of the DNA (Desiderio and Kelly 1981). The TP is synthesized as an 80K precursor (pTP) which is processed late in infection to the 55K form by an Ad-coded protease (Challberg and Kelly 1981; Stillman 1981; Lichy et al. 1982 b). Mapping of the TP by in vitro translation of mRNAs hybridized to restriction fragments of Ad DNA (Stillman et al. 1981) showed it to be a viral gene product encoded between map coordinates 11 and 30 in the E2B region. Subsequent determination of the DNA sequence of this region (Alestrom et al. 1982), together with partial amino acid sequence studies of the TP (Smart and Stillman 1982) further localized the TP gene to an open reading frame between coordinates 28.4 and 23.2 on the leftward strand.
KeywordsNuclear Extract Terminal Protein Glycerol Gradient Complete Reaction Mixture Crude Nuclear Extract
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