Construction of a cDNA library from polyadenylated RNA of Bacillus subtilis and the determination of some 3′-terminal sequences

Abstract

We had found previously that polyadenylated RNA constitutes a surprisingly large fraction of mRNA m both Escherichia coli and Bacillus subtilis [Gopalakrishna et al., Nucl. Acids Res. 9 (1981) 3545–3554; Biochem. 21 (1982) 2724–2729]. We have also shown [Gopalakrishna and Sarkar, J. Biol. Chem. 257 (1982) 2747–2750] that polyadenylated RNA from B. subtilis can serve as a template for the synthesis of complementary DNA by reverse transcriptase using oligo(dT) as primer. In this work, we show that the cDNA thus synthesized contains sequences representative of poly(A)+ RNA and can serve as template for double-stranded (ds) cDNA synthesis. The ds cDNA could be inserted into the PstI site of pBR322 and cloned in E. coli DH1. The cDNA inserts from a few cloned recombinant pBR322 plasmids were transferred to M13mp18 bacteriophage for sequence determination. Six cDNA species had terminal oligo(dT) sequences, indicating that they represented the complement of poly(A)+ RNA. This constitutes independent and direct evidence for the existence of bacterial polyadenylated mRNA and opens the way for studying the nucleotide sequences that control polyadenylation.