A NEW APPROACH TO DETERMINATION OF NUCLEOTIDE SEQUENCE OF A VIRAL RNA

A Qβ RNA segment was produced in vitro by means of a synchronized minus‐strand‐directed synthesis using purified Qβ replicase, a reaction known to yield infectious Qβ RNA. Arguments are presented that a 5.5S product obtained by incubation for 30 seconds at 20°C corresponds to the 5′‐terminal region of Qβ RNA. After obtaining and sequencing 26 T1 and 21 pancreatic RNase products, we established 3′ nearest neighbors by using products labeled with only one nucleoside [α‐32P]triphosphate at a time. The ordering of the oligonucleotides was facilitated by observing the time of their appearance in products from incubations of 5, 10, 15, 20, and 25 seconds. The enzymatic product consists of 160 nucleotides; no known initiation triplet appears before the sixtieth nucleotide. There is no indication that the coat protein cistron starts within the RNA segment analyzed. Copyright © 1969 Wiley‐Liss, Inc.