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. 1980 Jul 1;86(1):235–243. doi: 10.1083/jcb.86.1.235

Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes

PMCID: PMC2110653  PMID: 6998985

Abstract

Antibodies were raised in chickens against heterogeneous nuclear RNA (hnRNA)-binding proteins from 30S ribonucleoprotein (RNP) complexes of mouse Taper hepatoma ascites cell nuclei. The antibody preparations were characterized for immunological specificity and purity by double- diffusion gels, binding to specific bands in SDS polyacrylamide gels, and crossed immunoelectrophoresis. Antibodies raised against either whole 30S RNP complexes or purified RNP core proteins had a strong selective affinity for the four 34,000- to 40,000-dalton polypeptides which comprise the major structural proteins of hnRNP. The intracellular distribution of 30S RNP antigens in mouse ascites cells was determined by indirect immunofluorescence microsacopy. In interphase cells immunofluorescent sites were restricted to the nucleus, and nucleoli were free of fluorescence. The chicken anti-mouse- RNP antibodies were also able to react with cells from many different vertebrate species, showing a similar nucleus-restricted localization of the reacting sites. The antibodies also bound chick 30S RNP-proteins and reacted with the nuclei of chick cells. An exception to this was the failure of the antibody to bind to adult chick erythrocytes, suggesting that these major hnRNA binding proteins may be found only in nuclei capable of RNA synthesis.

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Selected References

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

  1. Axén R., Porath J., Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature. 1967 Jun 24;214(5095):1302–1304. doi: 10.1038/2141302a0. [DOI] [PubMed] [Google Scholar]
  2. Beyer A. L., Christensen M. E., Walker B. W., LeStourgeon W. M. Identification and characterization of the packaging proteins of core 40S hnRNP particles. Cell. 1977 May;11(1):127–138. doi: 10.1016/0092-8674(77)90323-3. [DOI] [PubMed] [Google Scholar]
  3. Billings P. B., Martin T. E. Proteins of nuclear ribonucleoprotein subcomplexes. Methods Cell Biol. 1978;17:349–376. doi: 10.1016/s0091-679x(08)61154-1. [DOI] [PubMed] [Google Scholar]
  4. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burridge K. Changes in cellular glycoproteins after transformation: identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4457–4461. doi: 10.1073/pnas.73.12.4457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burridge K. Direct identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Methods Enzymol. 1978;50:54–64. doi: 10.1016/0076-6879(78)50007-4. [DOI] [PubMed] [Google Scholar]
  7. Cort S., McDougal J. S. Isolation, lactoperoxidase catalyzed radioiodination, and recovery of proteins bound to insoluble immunoadsorbents. J Immunol Methods. 1977;18(3-4):269–280. doi: 10.1016/0022-1759(77)90181-8. [DOI] [PubMed] [Google Scholar]
  8. Cuatrecasas P. Affinity chromatography. Annu Rev Biochem. 1971;40:259–278. doi: 10.1146/annurev.bi.40.070171.001355. [DOI] [PubMed] [Google Scholar]
  9. Cuatrecasas P., Wilchek M., Anfinsen C. B. Selective enzyme purification by affinity chromatography. Proc Natl Acad Sci U S A. 1968 Oct;61(2):636–643. doi: 10.1073/pnas.61.2.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. GOODMAN M., WOLFE H. R., NORTON S. Precipitin production in chickens. VI. The effect of varying concentrations of NaCl on precipitate formation. J Immunol. 1951 Feb;66(2):225–236. [PubMed] [Google Scholar]
  11. Granger B. L., Lazarides E. The existence of an insoluble Z disc scaffold in chicken skeletal muscle. Cell. 1978 Dec;15(4):1253–1268. doi: 10.1016/0092-8674(78)90051-x. [DOI] [PubMed] [Google Scholar]
  12. Hersh R. T., Benedict A. A. Aggregation of chicken gamma-G immunoglobulin in 1.5 M sodium chloride solution. Biochim Biophys Acta. 1966 Jan 25;115(1):242–244. doi: 10.1016/0304-4165(66)90074-2. [DOI] [PubMed] [Google Scholar]
  13. Kinniburgh A. J., Billings P. B., Quinlan T. J., Martin T. E. Distribution of hnRNA and mRNA sequences in nuclear ribonucleoprotein complexes. Prog Nucleic Acid Res Mol Biol. 1976;19:335–351. doi: 10.1016/s0079-6603(08)60930-7. [DOI] [PubMed] [Google Scholar]
  14. Kumar A., Warner J. R. Characterization of ribosomal precursor particles from HeLa cell nucleoli. J Mol Biol. 1972 Jan 28;63(2):233–246. doi: 10.1016/0022-2836(72)90372-5. [DOI] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. 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]
  17. Lukanidin E. M., Olsnes S., Pihl A. Antigenic difference between informofers and protein bound to polyribosomal mRNA from rat liver. Nat New Biol. 1972 Nov 15;240(98):90–92. doi: 10.1038/newbio240090a0. [DOI] [PubMed] [Google Scholar]
  18. Marchalonis J. J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem J. 1969 Jun;113(2):299–305. doi: 10.1042/bj1130299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Martin T. E., McCarthy B. J. Synthesis and turnover of RNA in the 30-S nuclear ribonucleoprotein complexes of mouse ascites cells. Biochim Biophys Acta. 1972 Aug 25;277(2):354–367. doi: 10.1016/0005-2787(72)90417-0. [DOI] [PubMed] [Google Scholar]
  20. Martin T., Billings P., Levey A., Ozarslan S., Quinlan T., Swift H., Urbas L. Some properties of RNA:protein complexes from the nucleus of eukaryotic cells. Cold Spring Harb Symp Quant Biol. 1974;38:921–932. doi: 10.1101/sqb.1974.038.01.094. [DOI] [PubMed] [Google Scholar]
  21. Martin T., Billings P., Pullman J., Stevens B., Kinniburgh A. Substructure of nuclear ribonucleoprotein complexes. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):899–909. doi: 10.1101/sqb.1978.042.01.091. [DOI] [PubMed] [Google Scholar]
  22. Miller O. L., Jr, Bakken A. H. Morphological studies of transcription. Acta Endocrinol Suppl (Copenh) 1972;168:155–177. doi: 10.1530/acta.0.071s155. [DOI] [PubMed] [Google Scholar]
  23. Olden K., Yamada K. M. Direct detection of antigens in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1977 Apr;78(2):483–490. doi: 10.1016/0003-2697(77)90108-7. [DOI] [PubMed] [Google Scholar]
  24. Palacios R., Palmiter R. D., Schimke R. T. Identification and isolation of ovalbumin-synthesizing polysomes. I. Specific binding of 125 I-anti-ovalbumin to polysomes. J Biol Chem. 1972 Apr 25;247(8):2316–2321. [PubMed] [Google Scholar]
  25. Pederson T. Proteins associated with heterogeneous nuclear RNA in eukaryotic cells. J Mol Biol. 1974 Feb 25;83(2):163–183. doi: 10.1016/0022-2836(74)90386-6. [DOI] [PubMed] [Google Scholar]
  26. Rodman T. C., Litwin S. D., Romani M., Vidali G. Life history of mouse sperm protein. Intratesticular stages. J Cell Biol. 1979 Mar;80(3):605–620. doi: 10.1083/jcb.80.3.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Samarina O. P., Krichevskaya A. A., Georgiev G. P. Nuclear ribonucleoprotein particles containing messenger ribonucleic acid. Nature. 1966 Jun 25;210(5043):1319–1322. doi: 10.1038/2101319a0. [DOI] [PubMed] [Google Scholar]
  28. Samarina O. P., Lukanidin E. M., Molnar J., Georgiev G. P. Structural organization of nuclear complexes containing DNA-like RNA. J Mol Biol. 1968 Apr 14;33(1):251–263. doi: 10.1016/0022-2836(68)90292-1. [DOI] [PubMed] [Google Scholar]
  29. Silver L. M., Wu C. E., Elgin S. C. Immunofluorescent techniques in the analysis of chromosomal proteins. Methods Cell Biol. 1978;18:151–167. doi: 10.1016/s0091-679x(08)60138-7. [DOI] [PubMed] [Google Scholar]
  30. Taper H. S., Woolley G. W., Teller M. N., Lardis M. P. A new transplantable mouse liver tumor of spontaneous origin. Cancer Res. 1966 Jan;26(1):143–148. [PubMed] [Google Scholar]
  31. Weeke B. Crossed immunoelectrophoresis. Scand J Immunol Suppl. 1973;1:47–56. doi: 10.1111/j.1365-3083.1973.tb03778.x. [DOI] [PubMed] [Google Scholar]

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