Skip to main content
PMC 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
. 1982 Jul;79(13):4015–4019. doi: 10.1073/pnas.79.13.4015

Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.

J P Kraus, L E Rosenberg
PMCID: PMC346567  PMID: 6180433

Abstract

We have purified three low-abundance hepatic mRNAs to near homogeneity by polysome immunoadsorption. The mRNAs coding for the precursor of ornithine transcarbamoylase [carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3], the precursor of the beta-subunit of propionyl-CoA carboxylase [propionyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3], and cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], representing approximately 0.20, 0.02, and 0.015% of total hepatic mRNA, respectively, were purified 450- to 6,300-fold. We used the following steps: interaction of rat liver polysomes with an IgG fraction of monospecific antisera raised against each polypeptide; immobilization of polysome-antibody complexes on a protein A-Sepharose column; removal of the bulk of polysomes by extensive washing; dissociation of ribosomal subunits and elution of specific mRNA with EDTA; and isolation of the eluted mRNA by chromatography on an oligo(dT)-cellulose column. It seems likely that this procedure will permit isolation of other low-abundance mRNAs and subsequent cloning of their respective cDNAs.

Full text

PDF
4017

Images in this article

4017Image
on p.4017
4017Image
on p.4017
4018Image
on p.4018

Selected References

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

  1. Brown D. D., Suzuki Y. The purification of the messenger RNA for silk fibroin. Methods Enzymol. 1974;30:648–654. doi: 10.1016/0076-6879(74)30062-6. [DOI] [PubMed] [Google Scholar]
  2. COWIE D. B., SPIEGELMAN S., ROBERTS R. B., DUERKSEN J. D. Ribosome-bound beta-galactosidase. Proc Natl Acad Sci U S A. 1961 Jan 15;47:114–122. doi: 10.1073/pnas.47.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Conboy J. G., Kalousek F., Rosenberg L. E. In vitro synthesis of a putative precursor of mitochondrial ornithine transcarbamoylase. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5724–5727. doi: 10.1073/pnas.76.11.5724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Deeley R. G., Gordon J. I., Burns A. T., Mullinix K. P., Binastein M., Goldberg R. F. Primary activation of the vitellogenin gene in the rooster. J Biol Chem. 1977 Nov 25;252(22):8310–8319. [PubMed] [Google Scholar]
  5. Derynck R., Remaut E., Saman E., Stanssens P., De Clercq E., Content J., Fiers W. Expression of human fibroblast interferon gene in Escherichia coli. Nature. 1980 Sep 18;287(5779):193–197. doi: 10.1038/287193a0. [DOI] [PubMed] [Google Scholar]
  6. Dugaiczyk A., Law S. W., Dennison O. E. Nucleotide sequence and the encoded amino acids of human serum albumin mRNA. Proc Natl Acad Sci U S A. 1982 Jan;79(1):71–75. doi: 10.1073/pnas.79.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Flick P. K., Chen J., Alberts A. W., Vagelos P. R. Translation of rat liver fatty acid synthetase mRNA in a cell-free system derived from wheat germ. Proc Natl Acad Sci U S A. 1978 Feb;75(2):730–734. doi: 10.1073/pnas.75.2.730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gough N. M., Adams J. M. Immunoprecipitation of specific polysomes using Staphylococcus aureus: purification of the immunoglobulin k chain messenger RNA from the mouse myeloma MPC11. Biochemistry. 1978 Dec 12;17(25):5560–5566. doi: 10.1021/bi00618a036. [DOI] [PubMed] [Google Scholar]
  9. Gravel R. A., Lam K. F., Mahuran D., Kronis A. Purification of human liver propionyl-CoA carboxylase by carbon tetrachloride extraction and monomeric avidin affinity chromatography. Arch Biochem Biophys. 1980 May;201(2):669–673. doi: 10.1016/0003-9861(80)90557-3. [DOI] [PubMed] [Google Scholar]
  10. Groner B., Hynes N. E., Sippel A. E., Jeep S., Chi-Nguyen-Huu M., Schütz G. Immunoadsorption of specific chicken oviduct polysomes. Isolation of ovalbumin, ovomucoid, and lysozyme messenger RNA. J Biol Chem. 1977 Oct 10;252(19):6666–6674. [PubMed] [Google Scholar]
  11. Hoogenraad N. J., Sutherland T. M., Howlett G. J. Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog. Anal Biochem. 1980 Jan 1;101(1):97–102. doi: 10.1016/0003-2697(80)90045-7. [DOI] [PubMed] [Google Scholar]
  12. Korman A. J., Knudsen P. J., Kaufman J. F., Strominger J. L. cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1844–1848. doi: 10.1073/pnas.79.6.1844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kraus J. P., Conboy J. G., Rosenberg L. E. Pre-ornithine transcarbamylase. Properties of the cytoplasmic precursor of a mitochondrial matrix enzyme. J Biol Chem. 1981 Nov 10;256(21):10739–10742. [PubMed] [Google Scholar]
  14. Kraus J., Packman S., Fowler B., Rosenberg L. E. Purification and properties of cystathionine beta-synthase from human liver. Evidence for identical subunits. J Biol Chem. 1978 Sep 25;253(18):6523–6528. [PubMed] [Google Scholar]
  15. 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]
  16. Lee D. C., McKnight G. S., Palmiter R. D. The action of estrogen and progesterone on the expression of the transferrin gene. A comparison of the response in chick liver and oviduct. J Biol Chem. 1978 May 25;253(10):3494–3503. [PubMed] [Google Scholar]
  17. Lheureux M., Lheureux M., Vervoort T., Van Meirvenne N., Steinert M. Immunological purification and partial characterization of variant-specific surface antigen messenger RNA of Trypanosoma brucei brucei. Nucleic Acids Res. 1979 Oct 10;7(3):595–609. doi: 10.1093/nar/7.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maurer R. A. Immunochemical isolation of prolactin messenger RNA. J Biol Chem. 1980 Feb 10;255(3):854–859. [PubMed] [Google Scholar]
  19. Mueller-Lantzsch N., Fan H. Monospecific immunoprecipitation of murine leukemia virus polyribosomes: identification of p30 protein-specific messenger RNA. Cell. 1976 Dec;9(4 Pt 1):579–588. doi: 10.1016/0092-8674(76)90040-4. [DOI] [PubMed] [Google Scholar]
  20. Nienhuis A. W., Falvey A. K., Anderson W. F. Preparation of globin messenger RNA. Methods Enzymol. 1974;30:621–630. doi: 10.1016/0076-6879(74)30060-2. [DOI] [PubMed] [Google Scholar]
  21. Palacios R., Sullivan D., Summers N. M., Kiely M. L., Schimke R. T. Purification of ovalbumin messenger ribonucleic acid by specific immunoadsorption of ovalbumin-synthesizing polysomes and millipore partition of ribonucleic acid. J Biol Chem. 1973 Jan 25;248(2):540–548. [PubMed] [Google Scholar]
  22. Parnes J. R., Velan B., Felsenfeld A., Ramanathan L., Ferrini U., Appella E., Seidman J. G. Mouse beta 2-microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2253–2257. doi: 10.1073/pnas.78.4.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Payvar F., Schimke R. T. Improvements in immunoprecipitation of specific messenger RNA. Isolation of highly purified conalbumin mRNA in high yield. Eur J Biochem. 1979 Nov 1;101(1):271–282. doi: 10.1111/j.1432-1033.1979.tb04240.x. [DOI] [PubMed] [Google Scholar]
  24. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schechter I. Use of antibodies for the isolation of biologically pure messenger ribonucleic acid from fully functional eukaryotic cells. Biochemistry. 1974 Apr 23;13(9):1875–1885. doi: 10.1021/bi00706a016. [DOI] [PubMed] [Google Scholar]
  26. Schutz G., Kieval S., Groner B., Sippel A. E., Kurtz D., Feigelson P. Isolation of specific messenger RNA by adsorption of polysomes to matrix-bound antibody. Nucleic Acids Res. 1977 Jan;4(1):71–84. doi: 10.1093/nar/4.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shapiro D. J., Taylor J. M., McKnight G. S., Palacios R., Gonzalez C., Kiely M. L., Schimke R. T. Isolation of hen oviduct ovalbumin and rat live albumin polysomes by indirect immunoprecipitation. J Biol Chem. 1974 Jun 25;249(12):3665–3671. [PubMed] [Google Scholar]
  28. Shapiro S. Z., Young J. R. An immunochemical method for mRNA purification. Application to messenger RNA encoding trypanosome variable surface antigen. J Biol Chem. 1981 Feb 25;256(4):1495–1498. [PubMed] [Google Scholar]
  29. Sidorova E. V., Trudolyubova M. G., Lerman M. I. A general method for isolation of individual poly-ribosomes and pure mRNAs by immunosorption technique. Mol Biol Rep. 1974 Sep;1(7):401–408. doi: 10.1007/BF00385673. [DOI] [PubMed] [Google Scholar]
  30. 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]

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