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. 1981 Jul 24;9(14):3545–3554. doi: 10.1093/nar/9.14.3545

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure.

Y Gopalakrishna, D Langley, N Sarkar
PMCID: PMC327370  PMID: 6169008

Abstract

A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described. Pulse-labeled RNA isolated by this procedure for Bacillus brevis, Bacillus subtilis, and Escherichia coli B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo(dT)-cellulose. This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA. The presence of polyadenylated RNA in E. coli was confirmed by an independent method which involved hybridization with [3H]polyuridylic acid. Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-treated cells of B. brevis, B. subtilis, and E. coli.

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Selected References

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  1. ARBER W. HOST SPECIFICITY OF DNA PRODUCED BY ESCHERICHIA COLI V . THE ROLE OF METHIONINE IN THE PRODUCTION OF HOST SPECIFICITY. J Mol Biol. 1965 Feb;11:247–256. doi: 10.1016/s0022-2836(65)80055-9. [DOI] [PubMed] [Google Scholar]
  2. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bergmann I. E., Brawerman G. Loss of the polyadenylate segment from mammalian messenger RNA. Selective cleavage of this sequence from polyribosomes. J Mol Biol. 1980 May 25;139(3):439–454. doi: 10.1016/0022-2836(80)90140-0. [DOI] [PubMed] [Google Scholar]
  4. Bina M., Feldmann R. J., Deeley R. G. Could poly(A) align the splicing sites of messenger RNA precursors? Proc Natl Acad Sci U S A. 1980 Mar;77(3):1278–1282. doi: 10.1073/pnas.77.3.1278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brawerman G. Characteristics and significance of the polyadenylate sequence in mammalian messenger RNA. Prog Nucleic Acid Res Mol Biol. 1976;17:117–148. doi: 10.1016/s0079-6603(08)60068-9. [DOI] [PubMed] [Google Scholar]
  6. Darnell J. E., Wall R., Tushinski R. J. An adenylic acid-rich sequence in messenger RNA of HeLa cells and its possible relationship to reiterated sites in DNA. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1321–1325. doi: 10.1073/pnas.68.6.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deutscher M. P. Synthesis and degradation of poly(A) in permeable cells of Escherichia coli. J Biol Chem. 1978 Aug 25;253(16):5579–5584. [PubMed] [Google Scholar]
  8. Edmonds M., Vaughan M. H., Jr, Nakazato H. Polyadenylic acid sequences in the heterogeneous nuclear RNA and rapidly-labeled polyribosomal RNA of HeLa cells: possible evidence for a precursor relationship. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1336–1340. doi: 10.1073/pnas.68.6.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geiduschek E. P., Haselkorn R. Messenger RNA. Annu Rev Biochem. 1969;38:647–676. doi: 10.1146/annurev.bi.38.070169.003243. [DOI] [PubMed] [Google Scholar]
  10. Kaur S., Jayaraman K. Appearance of polyadenylated RNA species during sporulation in Bacillus polymyxa. Biochem Biophys Res Commun. 1979 Jan 30;86(2):331–339. doi: 10.1016/0006-291x(79)90870-2. [DOI] [PubMed] [Google Scholar]
  11. Kerjan P., Szulmajster J. Isolation and characterization of polyadenylated RNA species from sporulating cells of Bacillus subtilis. Biochem Biophys Res Commun. 1980 Mar 13;93(1):201–208. doi: 10.1016/s0006-291x(80)80266-x. [DOI] [PubMed] [Google Scholar]
  12. Lee S. Y., Mendecki J., Brawerman G. A polynucleotide segment rich in adenylic acid in the rapidly-labeled polyribosomal RNA component of mouse sarcoma 180 ascites cells. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1331–1335. doi: 10.1073/pnas.68.6.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. MCCARTHY B. J., BOLTON E. T. INTERACTION OF COMPLEMENTARY RNA AND DNA. J Mol Biol. 1964 Feb;8:184–200. doi: 10.1016/s0022-2836(64)80128-5. [DOI] [PubMed] [Google Scholar]
  14. Mendelsohn S. L., Young D. A. Inhibition of ribonuclease. Efficacy of sodium dodecyl sulfate, diethyl pyrocarbonate, protein ase K and heparin using a sensitive ribonuclease assay. Biochim Biophys Acta. 1978 Jul 24;519(2):461–473. doi: 10.1016/0005-2787(78)90099-0. [DOI] [PubMed] [Google Scholar]
  15. Nakazato H., Venkatesan S., Edmonds M. Polyadenylic acid sequences in E. coli messenger RNA. Nature. 1975 Jul 10;256(5513):144–146. doi: 10.1038/256144a0. [DOI] [PubMed] [Google Scholar]
  16. Nudel U., Soreq H., Littauer U. Z. Globin mRNA species containing poly(A) segments of different lengths. Their functional stability in Xenopus oocytes. Eur J Biochem. 1976 Apr 15;64(1):115–121. doi: 10.1111/j.1432-1033.1976.tb10279.x. [DOI] [PubMed] [Google Scholar]
  17. Ohta N., Sanders M., Newton A. Characterization of unstable poly (A)-RNA in Caulobacter crescentus. Biochim Biophys Acta. 1978 Jan 26;517(1):65–75. doi: 10.1016/0005-2787(78)90034-5. [DOI] [PubMed] [Google Scholar]
  18. Reiner A. M. Characterization of polynucleotide phosphorylase mutants of Escherichia coli. J Bacteriol. 1969 Mar;97(3):1437–1443. doi: 10.1128/jb.97.3.1437-1443.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sarkar N., Langley D., Paulus H. Isolation and characterization of polyadenylate-containing RNA from Bacillus brevis. Biochemistry. 1978 Aug 22;17(17):3468–3474. doi: 10.1021/bi00610a007. [DOI] [PubMed] [Google Scholar]
  20. Sarkar N., Paulus H. A guanosine 3':5'-monophosphate-sensitive nuclease from Bacillus brevis. J Biol Chem. 1975 Jan 25;250(2):684–690. [PubMed] [Google Scholar]
  21. Schultz G. A., Chaconas G., Moore R. L. Polyadenylic acid sequences in the RNA of Hyphomicrobium. J Bacteriol. 1978 Feb;133(2):569–575. doi: 10.1128/jb.133.2.569-575.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Srinivasan P. R., Ramanarayanan M., Rabbani E. Presence of polyriboadenylate sequences in pulse-labeled RNA of Escherichia coli. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2910–2914. doi: 10.1073/pnas.72.8.2910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wiegers U., Hilz H. Rapid isolation of undegraded polysomal RNA without phenol. FEBS Lett. 1972 Jun 1;23(1):77–82. doi: 10.1016/0014-5793(72)80289-8. [DOI] [PubMed] [Google Scholar]
  24. Williams D. L., Klett H. Measurement of polyadenylic acid by hybridization with polyuridylic acid: a source of error due to the lability of tritiated polyuridylic acid in trichloroacetic acid. Anal Biochem. 1978 Nov;91(1):173–179. doi: 10.1016/0003-2697(78)90828-x. [DOI] [PubMed] [Google Scholar]

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