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. 1986 Aug 26;14(16):6433–6451. doi: 10.1093/nar/14.16.6433

The use of single-stranded DNA and RNase H to promote quantitative 'hybrid arrest of translation' of mRNA/DNA hybrids in reticulocyte lysate cell-free translations.

J Minshull, T Hunt
PMCID: PMC311656  PMID: 3018671

Abstract

Single-stranded cDNA clones complementary to the 5' end of TMV RNA have been used to explore the conditions necessary for efficient 'hybrid arrest of translation' in the reticulocyte lysate. It is shown that incubations of 20 minutes at 60 degrees in 0.1 M KCl are sufficient to give almost complete arrest of translation using a clone complementary to the 5'-non-coding region and first 171 coding nucleotides of TMV RNA. However, hybrids with DNA complementary to regions of the mRNA downstream of the first AUG gave variable and in some cases almost no arrest of translation in the reticulocyte lysate unless they were first digested with RNase H. A simple and rapid method for giving complete and highly specific arrest of translation of particular mRNAs in complex mixtures has been developed using both cDNA clones and synthetic oligodeoxynucleotides in conjunction with RNase H digestion. Evidence is presented that suggests that 'hybrid arrest of translation' in the wheat-germ cell-free system is primarily due to the action of RNase H. When a reticulocyte lysate was doped with 20 U/ml of RNase H, its ability to translate unannealed mRNA was unaffected but it translated DNA/RNA hybrids extremely poorly.

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Selected References

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

  1. 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]
  2. Blake K. R., Murakami A., Miller P. S. Inhibition of rabbit globin mRNA translation by sequence-specific oligodeoxyribonucleotides. Biochemistry. 1985 Oct 22;24(22):6132–6138. doi: 10.1021/bi00343a015. [DOI] [PubMed] [Google Scholar]
  3. Blakesley R. W., Wells R. D. 'Single-stranded' DNA from phiX174 and M13 is cleaved by certain restriction endonucleases. Nature. 1975 Oct 2;257(5525):421–422. doi: 10.1038/257421a0. [DOI] [PubMed] [Google Scholar]
  4. Chandler P. M. The use of single-stranded phage DNAs in hybrid arrest and release translation. Anal Biochem. 1982 Nov 15;127(1):9–16. doi: 10.1016/0003-2697(82)90137-3. [DOI] [PubMed] [Google Scholar]
  5. Donis-Keller H. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 1979 Sep 11;7(1):179–192. doi: 10.1093/nar/7.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Erickson A. H., Blobel G. Cell-free translation of messenger RNA in a wheat germ system. Methods Enzymol. 1983;96:38–50. doi: 10.1016/s0076-6879(83)96007-x. [DOI] [PubMed] [Google Scholar]
  7. Evans T., Rosenthal E. T., Youngblom J., Distel D., Hunt T. Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell. 1983 Jun;33(2):389–396. doi: 10.1016/0092-8674(83)90420-8. [DOI] [PubMed] [Google Scholar]
  8. Goelet P., Karn J. Methods for cDNA cloning and sequencing tobacco mosaic virus RNA. Gene. 1984 Sep;29(3):331–342. doi: 10.1016/0378-1119(84)90062-3. [DOI] [PubMed] [Google Scholar]
  9. Goelet P., Lomonossoff G. P., Butler P. J., Akam M. E., Gait M. J., Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5818–5822. doi: 10.1073/pnas.79.19.5818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haeuptle M. T., Frank R., Dobberstein B. Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length. Nucleic Acids Res. 1986 Feb 11;14(3):1427–1448. doi: 10.1093/nar/14.3.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harland R., Weintraub H. Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA. J Cell Biol. 1985 Sep;101(3):1094–1099. doi: 10.1083/jcb.101.3.1094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hausen P., Stein H. Ribonuclease H. An enzyme degrading the RNA moiety of DNA-RNA hybrids. Eur J Biochem. 1970 Jun;14(2):278–283. doi: 10.1111/j.1432-1033.1970.tb00287.x. [DOI] [PubMed] [Google Scholar]
  13. Jackson R. J., Hunt T. Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 1983;96:50–74. doi: 10.1016/s0076-6879(83)96008-1. [DOI] [PubMed] [Google Scholar]
  14. Kawasaki E. S. Quantitative hybridization-arrest of mRNA in Xenopus oocytes using single-stranded complementary DNA or oligonucleotide probes. Nucleic Acids Res. 1985 Jul 11;13(13):4991–5004. doi: 10.1093/nar/13.13.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krainer A. R., Maniatis T. Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. Cell. 1985 Oct;42(3):725–736. doi: 10.1016/0092-8674(85)90269-7. [DOI] [PubMed] [Google Scholar]
  16. Krämer A., Keller W., Appel B., Lührmann R. The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors. Cell. 1984 Aug;38(1):299–307. doi: 10.1016/0092-8674(84)90551-8. [DOI] [PubMed] [Google Scholar]
  17. Liebhaber S. A., Cash F. E., Shakin S. H. Translationally associated helix-destabilizing activity in rabbit reticulocyte lysate. J Biol Chem. 1984 Dec 25;259(24):15597–15602. [PubMed] [Google Scholar]
  18. Melton D. A. Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci U S A. 1985 Jan;82(1):144–148. doi: 10.1073/pnas.82.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  20. Miller J. S., Paterson B. M., Ricciardi R. P., Cohen L., Roberts B. E. Methods utilizing cell-free protein-synthesizing systems for the identification of recombinant DNA molecules. Methods Enzymol. 1983;101:650–674. doi: 10.1016/0076-6879(83)01046-0. [DOI] [PubMed] [Google Scholar]
  21. Muthukrishnan S., Both G. W., Furuichi Y., Shatkin A. J. 5'-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation. Nature. 1975 May 1;255(5503):33–37. doi: 10.1038/255033a0. [DOI] [PubMed] [Google Scholar]
  22. Paterson B. M., Roberts B. E., Kuff E. L. Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4370–4374. doi: 10.1073/pnas.74.10.4370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Roth M. J., Tanese N., Goff S. P. Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. J Biol Chem. 1985 Aug 5;260(16):9326–9335. [PubMed] [Google Scholar]
  24. Sippel A. E., Stavrianopoulos J. G., Schutz G., Feigelson P. Translational properties of rabbit globin mRNA after specific removal of poly(A) with ribonuclease H. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4635–4639. doi: 10.1073/pnas.71.11.4635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Standart N. M., Bray S. J., George E. L., Hunt T., Ruderman J. V. The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs. J Cell Biol. 1985 Jun;100(6):1968–1976. doi: 10.1083/jcb.100.6.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wassarman P. M., Hollinger T. G., Smith L. D. Ribonuclease H activity in germinal vesicles of oocytes from Rana pipiens. Exp Cell Res. 1974 Dec;89(2):410–412. doi: 10.1016/0014-4827(74)90809-x. [DOI] [PubMed] [Google Scholar]

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