In vitro transcription of the inverted terminal repetition of the vaccinia virus genome: correspondence of initiation and cap sites.

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RESUMO

Specific RNAs synthesized in vitro by vaccinia virus cores were analyzed with the aid of DNA from the terminal 9,000 base pairs of the genome that was cloned in phage lambda, pBR322, and the single-stranded phage fl. Three mRNA's coding for polypeptides with molecular weights of 7,500 (7.5K), 19K, and 42K were shown to have sizes and map positions similar to those described for mRNA's made early in infection. A previously undescribed transcript made in vivo and in vitro, with a 5' end at about 8.7 kilobase pairs from the end of the genome, was also detected. After chemical removal of the terminal 7-methylguanosine residue, the 5' ends of the RNAs were specifically labeled by enzymatic capping and the mapped by gel electrophoresis of nuclease-resistant RNA.DNA hybrids, as well as by hybridization of the end-labeled RNA to immobilized DNA restriction fragments. Analysis of the purified cap structures demonstrated that three of the mRNA's have both m7G(5')pppAm and, m7G(5')pppGm ends, indicating some degree of terminal heterogeneity. The fourth transcript has exclusively m7G(5')pppAm ends. By synthesizing RNA in the presence of [beta-32P]GTP, it could be shown that cap sites correspond to sites of initiation of RNA synthesis.

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