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
. 1978 Mar;75(3):1217–1221. doi: 10.1073/pnas.75.3.1217

Analysis of mRNA populations by cDNA·mRNA hybrid-mediated inhibition of cell-free protein synthesis

Nicholas D Hastie 1, William A Held 1,*
PMCID: PMC411441  PMID: 274713

Abstract

Hybridization of mRNA to its corresponding cDNA was found to specifically inhibit translation of the mRNA in vitro. Using hybridization of globin cDNA to globin mRNA as a model system, we found that equivalent amounts of cDNA were required both for the saturation of the mRNA hybridization and for complete inhibition of globin synthesis. Also, the rate of inactivation of translation was identical to the rate of hybridization and followed the predicted kinetic form. This assay has been applied to the analysis of a set of abundant mRNAs in mouse liver. Hybridization of liver mRNA with total liver cDNA in slight excess to a low C0t value specifically inhibited translation of several major polypeptides. Melting of the hybrids prior to translation restored synthesis of these polypeptides. Moreover, we found that different liver mRNAs are inactivated with different kinetics; the results suggest that the mRNAs for the major urinary polypeptide and for albumin are the most abundant and second most abundant, respectively, in mouse liver. The general applications of this technique are discussed.

Keywords: globin mRNA, hybridization, translation, abundant liver mRNAs

Full text

PDF
1217

Images in this article

1218Image
on p.1218

Selected References

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

  1. Atkins J. F., Lewis J. B., Anderson C. W., Gesteland R. F. Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines. J Biol Chem. 1975 Jul 25;250(14):5688–5695. [PubMed] [Google Scholar]
  2. Bishop J. O., Rosbash M. Reiteration frequency of duck haemoglobin genes. Nat New Biol. 1973 Feb 14;241(111):204–207. doi: 10.1038/newbio241204a0. [DOI] [PubMed] [Google Scholar]
  3. Freienstein C., Blobel G. Use of eukaryotic native small ribosomal subunits for the translation of globin messenger RNA. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3435–3439. doi: 10.1073/pnas.71.9.3435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hastie N. D., Bishop J. O. The expression of three abundance classes of messenger RNA in mouse tissues. Cell. 1976 Dec;9(4 Pt 2):761–774. doi: 10.1016/0092-8674(76)90139-2. [DOI] [PubMed] [Google Scholar]
  5. Held W. A., West K., Gallagher J. F. Importance of initiation factor preparations in the translation of reovirus and globin mRNAs lacking a 5'-terminal 7-methylguanosine. J Biol Chem. 1977 Dec 10;252(23):8489–8497. [PubMed] [Google Scholar]
  6. Inglis S. C., McGeoch D. J., Mahy B. W. Polypeptides specified by the influenza virus genoma. 2. Assignement of protein coding functions to individual genome segments by in vitro translation. Virology. 1977 May 15;78(2):522–536. doi: 10.1016/0042-6822(77)90128-3. [DOI] [PubMed] [Google Scholar]
  7. Krystosek A., Cawthon M. L., Kabat D. Improved methods for purification and assay of eukaryotic messenger ribonucleic acids and ribosomes. Quantitative analysis of their interaction in a fractionated reticulocyte cell-free system. J Biol Chem. 1975 Aug 10;250(15):6077–6084. [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Paterson B. M., Bishop J. O. Changes in the mRNA population of chick myoblasts during myogenesis in vitro. Cell. 1977 Nov;12(3):751–765. doi: 10.1016/0092-8674(77)90275-6. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Schreier M. H., Staehelin T. Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems. J Mol Biol. 1973 Feb 19;73(3):329–349. doi: 10.1016/0022-2836(73)90346-x. [DOI] [PubMed] [Google Scholar]
  12. Taylor J. M., Tse T. P. Isolation of rat liver albumin messenger RNA. J Biol Chem. 1976 Dec 10;251(23):7461–7467. [PubMed] [Google Scholar]
  13. Vogt V. M. Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae. Eur J Biochem. 1973 Feb 15;33(1):192–200. doi: 10.1111/j.1432-1033.1973.tb02669.x. [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