Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1971 Sep;68(9):2268–2272. doi: 10.1073/pnas.68.9.2268

Purification and In Vitro Transcription of a Transfer RNA Gene

Violet Daniel *, Jacques S Beckmann *, Sara Sarid †, Jacob I Grimberg *, Max Herzberg ‡, Uriel Z Littauer *
PMCID: PMC389398  PMID: 4943795

Abstract

A gene specifying tyrosine transfer RNA has been purified and transcribed in vitro. The purification procedure made use of two specialized transducing phages carrying the tRNATyr gene of Escherichia coli inserted into their DNA in opposite orientations. The separated heavy strands of the two phages were annealed and the single-stranded tails of the resulting hybrid were removed by digestion with Neurospora endonuclease. The size of the purified double-stranded structures was determined by electron microscopy. These isolated duplexes served as template for the in vitro transcription of tRNATyr-like molecules.

Keywords: E. coli, bacteriophage, Neurospora endonuclease, tyrosine, electron microscopy

Full text

PDF
2268

Images in this article

2269Image
on p.2269
2270Image
on p.2270
2271Image
on p.2271

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andoh T., Ozeki H. Suppressor gene Su3+ of E. coli, a structural gene for tyrosine TRNA. Proc Natl Acad Sci U S A. 1968 Mar;59(3):792–799. doi: 10.1073/pnas.59.3.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brenner D. J., Fournier M. J., Doctor B. P. Isolation and partial characterization of the transfer ribonucleic acid cistrons from Escherichia coli. Nature. 1970 Aug 1;227(5257):448–451. doi: 10.1038/227448a0. [DOI] [PubMed] [Google Scholar]
  3. Brown D. D., Weber C. S. Gene linkage by RNA-DNA hybridization. I. Unique DNA sequences homologous to 4 s RNA, 5 s RNA and ribosomal RNA. J Mol Biol. 1968 Jun 28;34(3):661–680. doi: 10.1016/0022-2836(68)90188-5. [DOI] [PubMed] [Google Scholar]
  4. Burgess R. R. A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase. J Biol Chem. 1969 Nov 25;244(22):6160–6167. [PubMed] [Google Scholar]
  5. CHAMBERLIN M., BERG P. Deoxyribo ucleic acid-directed synthesis of ribonucleic acid by an enzyme from Escherichia coli. Proc Natl Acad Sci U S A. 1962 Jan 15;48:81–94. doi: 10.1073/pnas.48.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colli W., Oishi M. A procedure for gene purification: the purification of the ribosomal RNA genes of Bacillus subtilis as DNA-RNA hybrids. J Mol Biol. 1970 Aug;51(3):657–669. doi: 10.1016/0022-2836(70)90014-8. [DOI] [PubMed] [Google Scholar]
  7. Daniel V., Lavi S., Littauer U. Z. Isolation of biologically active transfer RNA from transfer RNA-DNA hybrids. Biochim Biophys Acta. 1969 May 20;182(1):76–84. doi: 10.1016/0005-2787(69)90522-x. [DOI] [PubMed] [Google Scholar]
  8. Daniel V., Sarid S., Beckmann J. S., Littauer U. Z. In vitro transcription of a transfer RNA gene. Proc Natl Acad Sci U S A. 1970 Aug;66(4):1260–1266. doi: 10.1073/pnas.66.4.1260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davis R. W., Davidson N. Electron-microscopic visualization of deletion mutations. Proc Natl Acad Sci U S A. 1968 May;60(1):243–250. doi: 10.1073/pnas.60.1.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jayaraman R., Goldberg E. B. A genetic assay for mRNA's of phage T4. Proc Natl Acad Sci U S A. 1969 Sep;64(1):198–204. doi: 10.1073/pnas.64.1.198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. KLEINSCHMIDT A. K., LANG D., JACHERTS D., ZAHN R. K. [Preparation and length measurements of the total desoxyribonucleic acid content of T2 bacteriophages]. Biochim Biophys Acta. 1962 Dec 31;61:857–864. [PubMed] [Google Scholar]
  12. Kohne D. E. Isolation and characterization of bacterial ribosomal RNA cistrons. Biophys J. 1968 Oct;8(10):1104–1118. doi: 10.1016/S0006-3495(68)86542-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LINN S., LEHMAN I. R. AN ENDONUCLEASE FROM NEUROSPORA CRASSA SPECIFIC FOR POLYNUCLEOTIDES LACKING AN ORDERED STRUCTURE. I. PURIFICATION AND PROPERTIES OF THE ENZYME. J Biol Chem. 1965 Mar;240:1287–1293. [PubMed] [Google Scholar]
  14. MATSUSHIRO A., SATO K., KIDA S. CHARACTERISTICS OF THE TRANSDUCING ELEMENTS OF BACTERIOPHAGE PHI-80. Virology. 1964 Jul;23:299–306. doi: 10.1016/0042-6822(64)90251-x. [DOI] [PubMed] [Google Scholar]
  15. Marks A., Spencer J. H. Isolation of Escherichia coli transfer RNA-gene hybrids. J Mol Biol. 1970 Jul 14;51(1):115–130. doi: 10.1016/0022-2836(70)90274-3. [DOI] [PubMed] [Google Scholar]
  16. Miller R. C., Jr, Besmer P., Khorana H. G., Fiandt M., Szybalski W. Studies on polynucleotides. XCVII. Opposing orientations and location of the su+3 gene in the transducing coliphages phi-80psu+3 and phi-80dsu+3su-3. J Mol Biol. 1971 Mar 14;56(2):363–368. doi: 10.1016/0022-2836(71)90470-0. [DOI] [PubMed] [Google Scholar]
  17. Rabin E. Z., Mustard M., Fraser M. J. Specific inhibition by ATP and other properites of an endonuclease of Neurospora crassa. Can J Biochem. 1968 Oct;46(10):1285–1291. doi: 10.1139/o68-193. [DOI] [PubMed] [Google Scholar]
  18. Russell R. L., Abelson J. N., Landy A., Gefter M. L., Brenner S., Smith J. D. Duplicate genes for tyrosine transfer RNA in Escherichia coli. J Mol Biol. 1970 Jan 14;47(1):1–13. doi: 10.1016/0022-2836(70)90397-9. [DOI] [PubMed] [Google Scholar]
  19. Ryan J. L., Morowitz H. J. Partial purification of native rRNA and tRNA cistrons from mycoplasma sp. (Kid). Proc Natl Acad Sci U S A. 1969 Aug;63(4):1282–1289. doi: 10.1073/pnas.63.4.1282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sgaramella V., Spadari S., Falaschi A. Isolation of the hybrid between ribosomal RNA and DNA of Bacillus subtilis. Cold Spring Harb Symp Quant Biol. 1968;33:839–842. doi: 10.1101/sqb.1968.033.01.095. [DOI] [PubMed] [Google Scholar]
  21. Shapiro J., Machattie L., Eron L., Ihler G., Ippen K., Beckwith J. Isolation of pure lac operon DNA. Nature. 1969 Nov 22;224(5221):768–774. doi: 10.1038/224768a0. [DOI] [PubMed] [Google Scholar]
  22. Takahashi H. An attempt to concentrate ribosomal RNA cistrons in Bacillus subtilis by Millipore filtration. Biochim Biophys Acta. 1969 Sep 17;190(1):214–216. doi: 10.1016/0005-2787(69)90171-3. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES