Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. II. Sequences of T1 and pancreatic ribonuclease digestion products of RNA transcripts prepared from subfragments of EcoRII-G.

The nucleotide sequences of pyrimidine tracts in subfragments produced by restriction endonuclease cleavage of the SV40 DNA fragment EcoRII-G was determined.Six subfragments were transcribed with Escherichia coli RNA polymerase in the presence of an cu-"2P-labeled nueleoside triphosphate.The sequence of the products obtained by extensive pancreatic and T, RNase digestion of these transcripts was deduced.The arguments used for the choice of the EcoRII-G' fragment of SV40 DNA to deduce the nucleotide sequence at the origin of DNA replication in this viral genome are presented in the preceding report (3).The restriction cleavage map of EcoRII-G and the strategy used for sequence analysis of this fragment are also described in the preceding communication (3).In brief, EcoRII-G was cleaved into smaller subfragments by use of various restriction endonucleases.The subfragments were transcribed with Escherichia coli RNA polymerase.The transcripts were digested with T, or pancreatic RNases and the products were fractionated, giving rise to characteristic oligonucleotide maps.Analysis of the sequences of these oligonucleotide products is presented in this report.EXPERIMENTAL PROCEDURES Materials Virus strains, radioactive compounds, and enzymes used were the same as described earlier (4, 5).EcoRII-G and subfragments thereof were prepared as described in the preceding report (3).Preparation of Labeled RNA Transcripts Intact form I SV40 DNA or fragments derived from it were transcribed by Escherichia coli RNA polymerasein the presence of all four ribonucleoside triphosphates, one of which was labeled with :IrP at the cz position in any one reaction.In a typical reaction, SV40 DNA was transcribed in a reaction mixture containing 1 to 3 pg of DNA, 1~1 ofRNA polymerase, 0.025 M TrislHCl, pH 7.9,0.05M KCI,