The synthesis of DNA complementary to polyadenylate-containing RNA from Bacillus subtilis.
We had shown earlier (Gopalakrishna, Y., Langley, D., Sarkar, N. (1981) Nucleic Acid Res. 9, 3545-3554) that a substantial fraction of mRNA of various bacterial species carries 3'-terminal polyadenylate sequences. In this paper, we show that poly(A)-containing RNA from Bacillus subtilis can serve as template for the synthesis of complementary DNA by avian myeloblastosis virus reverse transcriptase, provided that oligodeoxythymidylate is added as primer. Poly(A)-RNA purified by affinity chromatography on oligo(dT)-cellulose was 20 times more effective as template for cDNA formation than total bacterial RNA, whereas rRNA was inactive. The average chain length of the cDNA was 400 nucleotides (range = 230-800 nucleotides), and 95% of the cDNA could be degraded by the single-strand specific S1 nuclease after denaturation. The small fraction (5%) that was resistant to S1 nuclease may represent duplex hairpin structures. Annealing with poly(A)-RNA protected cDNA from degradation by S1 nuclease, indicating that cDNA indeed contains nucleotide sequences complementary to poly(A)-RNA. These results constitute independent evidence that a large fraction (about 40%) of B. subtilis mRNA is polyadenylated. Moreover, the synthesis of cDNA to bacterial mRNA provides an important new tool for the study of bacterial mRNA structure.
D001412:Bacillus subtilis / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics*; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D012333:RNA, Messenger; D013698:Templates, Genetic
D004269:DNA, Bacterial; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA