Isolation of ΦX174 Specific Messenger Ribonucleic Acids In Vivo and Identification of Their 5′ Terminal Nucleotides

Abstract

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) hybridization in formamide at low temperature was applied to hybridization of ΦX174 replicative form DNA and in vivo ΦX174 specific messenger RNA (mRNA) with some modification. We found that ΦX174 mRNA up to molecular weight 1.2 × 10 ⁶ could be hybridized to and eluted from DNA without apparent breakage of phosphodiester bonds and the 5′ terminal guanosine triphosphate and adenosine triphosphate of the RNA. By alkali hydrolysis of the purified in vivo ΦX174 mRNA and subsequent thin-layer chromatography of the digest, we isolated the 5′ terminal nucleotides and identified them as 2′- or 3′-monophosphate guanosine 5′-triphosphate (pppGp) and 2′- or 3′-monophosphate adenosine 5′-triphosphate (pppAp). By comparing the in vitro and in vivo synthesized ΦX174 mRNA, a difference in the pppAp-pppGp ratio was observed. In the in vitro RNA, this ratio was 1.5, whereas in the in vivo RNA it was 5.5.