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. 1981 Dec;1(12):1163–1176. doi: 10.1128/mcb.1.12.1163

Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

J Bruno, N Reich, J J Lucas
PMCID: PMC369742  PMID: 7346715

Abstract

The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed.

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Selected References

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

  1. Appels R., Williams A. F. Characterisation of RNA polymerase activity in avian red blood cells. Biochim Biophys Acta. 1970 Oct 15;217(2):531–534. doi: 10.1016/0005-2787(70)90551-4. [DOI] [PubMed] [Google Scholar]
  2. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  3. Brinster R. L., Chen H. Y., Trumbauer M. E., Avarbock M. R. Translation of globin messenger RNA by the mouse ovum. Nature. 1980 Jan 31;283(5746):499–501. doi: 10.1038/283499a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown J. L., Ingram V. M. Structural studies on chick embryonic hemoglobins. J Biol Chem. 1974 Jun 25;249(12):3960–3972. [PubMed] [Google Scholar]
  5. Bruns G. A., Ingram V. M. The erythroid cells and haemoglobins of the chick embryo. Philos Trans R Soc Lond B Biol Sci. 1973 Oct 25;266(877):225–305. doi: 10.1098/rstb.1973.0050. [DOI] [PubMed] [Google Scholar]
  6. Bunn H. F., McDonough M. Asymmetrical hemoglobin hybrids. An approach to the study of subunit interactions. Biochemistry. 1974 Feb 26;13(5):988–993. doi: 10.1021/bi00702a024. [DOI] [PubMed] [Google Scholar]
  7. CAMERON I. L., PRESCOTT D. M. RNA and protein metabolism in the maturation of the nucleated chicken erythrocyte. Exp Cell Res. 1963 May;30:609–612. doi: 10.1016/0014-4827(63)90344-6. [DOI] [PubMed] [Google Scholar]
  8. Campbell G. le M., Weintraub H., Mayall B. H., Holtzer H. Primitive erythropoiesis in early chick embryogenesis. II. Correlation between hemoglobin synthesis and the mitotic history. J Cell Biol. 1971 Sep;50(3):669–681. doi: 10.1083/jcb.50.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carlsson S. A., Moore G. P., Ringertz N. R. Nucleo-cytoplasmic protein migration during the activation of chick erythrocyte nuclei in heterokaryons. Exp Cell Res. 1973 Jan;76(1):234–241. doi: 10.1016/0014-4827(73)90441-2. [DOI] [PubMed] [Google Scholar]
  10. Carlsson S. A., Ringertz N. R., Savage R. E. Intracellular antigen migration in interspecific myoblast heterokaryons. Exp Cell Res. 1974 Mar 15;84(1):255–266. doi: 10.1016/0014-4827(74)90404-2. [DOI] [PubMed] [Google Scholar]
  11. Chapman B. S., Tobin A. J., Hood L. E. Complete amino acid sequence of the major early embryonic beta-like globin in chickens. J Biol Chem. 1981 Jun 10;256(11):5524–5531. [PubMed] [Google Scholar]
  12. Chapman B. S., Tobin A. J., Hood L. E. Complete amino acid sequences of the major early embryonic alpha-like globins of the chicken. J Biol Chem. 1980 Oct 10;255(19):9051–9059. [PubMed] [Google Scholar]
  13. Craig N. The effects of inhibitors of RNA and DNA synthesis on protein synthesis and polysome levels in mouse L-cells. J Cell Physiol. 1973 Oct;82(2):133–150. doi: 10.1002/jcp.1040820202. [DOI] [PubMed] [Google Scholar]
  14. Darzynkiewicz Z., Chelmicka-Szorc E., Arnason B. G. Suppressive effect of protease inhibitors on heterokaryons containing chick erythrocyte nuclei. Exp Cell Res. 1974 Aug;87(2):333–345. doi: 10.1016/0014-4827(74)90489-3. [DOI] [PubMed] [Google Scholar]
  15. Deacon N. J., Shine J., Naora H. Complete nucleotide sequence of a cloned chicken alpha-globin cDNA. Nucleic Acids Res. 1980 Mar 25;8(6):1187–1199. doi: 10.1093/nar/8.6.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dodgson J. B., Strommer J., Engel J. D. Isolation of the chicken beta-globin gene and a linked embryonic beta-like globin gene from a chicken DNA recombinant library. Cell. 1979 Aug;17(4):879–887. doi: 10.1016/0092-8674(79)90328-3. [DOI] [PubMed] [Google Scholar]
  17. Drysdale J. W., Righetti P., Bunn H. F. The separation of human and animal hemoglobins by isoelectric focusing in polyacrylamide gel. Biochim Biophys Acta. 1971 Jan 19;229(1):42–50. doi: 10.1016/0005-2795(71)90315-1. [DOI] [PubMed] [Google Scholar]
  18. Dupuy-Coin A. M., Ege T., Bouteille M., Ringertz N. R. Ultrastructure of chick erythrocyte nuclei undergoing reactivation in heterokaryons and enucleated cells. Exp Cell Res. 1976 Sep;101(2):355–369. doi: 10.1016/0014-4827(76)90388-8. [DOI] [PubMed] [Google Scholar]
  19. Ege T., Zeuthen J., Ringertz N. R. Reactivation of chick erythrocyte nuclei after fusion with enucleated cells. Somatic Cell Genet. 1975 Jan;1(1):65–80. doi: 10.1007/BF01538732. [DOI] [PubMed] [Google Scholar]
  20. Flint S. J., Weintraub H. M. An altered subunit configuration associated with the actively transcribed DNA of integrated adenovirus genes. Cell. 1977 Nov;12(3):783–794. doi: 10.1016/0092-8674(77)90277-x. [DOI] [PubMed] [Google Scholar]
  21. Garel A., Axel R. Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3966–3970. doi: 10.1073/pnas.73.11.3966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Garrels J. I. Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines. J Biol Chem. 1979 Aug 25;254(16):7961–7977. [PubMed] [Google Scholar]
  23. Groudine M., Holtzer H., Scherrer K., Therwath A. Lineage-dependent transcription of globin genes. Cell. 1974 Nov;3(3):243–247. doi: 10.1016/0092-8674(74)90138-x. [DOI] [PubMed] [Google Scholar]
  24. Groudine M., Peretz M., Weintraub H. Transcriptional regulation of hemoglobin switching in chicken embryos. Mol Cell Biol. 1981 Mar;1(3):281–288. doi: 10.1128/mcb.1.3.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. HELL A. THE INITIAL SYNTHESIS OF HAEMOGLOBIN IN DE-EMBRYONATED CHICK BLASTODERMS. I. METABOLISM OF THE BLASTODISC CULTURED IN VITRO. J Embryol Exp Morphol. 1964 Dec;12:609–619. [PubMed] [Google Scholar]
  26. Harris H., Sidebottom E., Grace D. M., Bramwell M. E. The expression of genetic information: a study with hybrid animal cells. J Cell Sci. 1969 Mar;4(2):499–525. doi: 10.1242/jcs.4.2.499. [DOI] [PubMed] [Google Scholar]
  27. Huez G., Bruck C., Cleuter Y. Translational stability of native and deadenylylated rabbit globin mRNA injected into HeLa cells. Proc Natl Acad Sci U S A. 1981 Feb;78(2):908–911. doi: 10.1073/pnas.78.2.908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Keane R. W., Abbott U. K. Erythropoiesis in normal and mutant chick embryos. Dev Biol. 1980 Mar 15;75(2):442–453. doi: 10.1016/0012-1606(80)90175-x. [DOI] [PubMed] [Google Scholar]
  29. Kernell A. M., Bolund L., Ringertz N. R. Chromatin changes during erythropoiesis. Exp Cell Res. 1971 Mar;65(1):1–6. doi: 10.1016/s0014-4827(71)80042-3. [DOI] [PubMed] [Google Scholar]
  30. Ladda R. L., Estensen R. D. Introduction of a heterologous nucleus into enucleated cytoplasms of cultured mouse L-cells. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1528–1533. doi: 10.1073/pnas.67.3.1528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lasky L., Nozick N. D., Tobin A. J. Few transcribed RNAs are translated in avian erythroid cells. Dev Biol. 1978 Nov;67(1):23–39. doi: 10.1016/0012-1606(78)90297-x. [DOI] [PubMed] [Google Scholar]
  32. Lasky L., Tobin A. J. Transcriptional regulation in avian erythroid cells. Biochemistry. 1979 Apr 17;18(8):1594–1598. doi: 10.1021/bi00575a033. [DOI] [PubMed] [Google Scholar]
  33. Laval M., Hernandez-Verdun D., Bouteille M. Remnant nucleolar structures and residual RNA synthesis in chick erythrocytes. Exp Cell Res. 1981 Mar;132(1):157–167. doi: 10.1016/0014-4827(81)90092-6. [DOI] [PubMed] [Google Scholar]
  34. Lipsich L. A., Lucas J. J., Kates J. R. Cell cycle dependence of the reactivation of chick erythrocyte nuclei after transplantation into mouse L929 cell cytoplasts. J Cell Physiol. 1978 Nov;97(2):199–207. doi: 10.1002/jcp.1040970209. [DOI] [PubMed] [Google Scholar]
  35. Longacre S. S., Rutter W. J. Isolation of chicken hemoglobin mRNA and synthesis of complementary DNA. J Biol Chem. 1977 Apr 25;252(8):2742–2752. [PubMed] [Google Scholar]
  36. Longacre S. S., Rutter W. J. Nucleotide polymerases in the developing avian erythrocyte. J Biol Chem. 1977 Jan 10;252(1):273–283. [PubMed] [Google Scholar]
  37. Lucas J. J., Ginsberg H. S. Transcription and transport of virus-specific ribonucleic acids in African green monkey kidney cells abortively infected with type 2 adenovirus. J Virol. 1972 Dec;10(6):1109–1117. doi: 10.1128/jvi.10.6.1109-1117.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lucas J. J., Kates J. R. Nuclear transplantation with mammalian cells. Methods Cell Biol. 1977;15:359–370. doi: 10.1016/s0091-679x(08)60225-3. [DOI] [PubMed] [Google Scholar]
  39. McGhee J. D., Ginder G. D. Specific DNA methylation sites in the vicinity of the chicken beta-globin genes. Nature. 1979 Aug 2;280(5721):419–420. doi: 10.1038/280419a0. [DOI] [PubMed] [Google Scholar]
  40. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  41. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  42. Reich E., Goldberg I. H. Actinomycin and nucleic acid function. Prog Nucleic Acid Res Mol Biol. 1964;3:183–234. doi: 10.1016/s0079-6603(08)60742-4. [DOI] [PubMed] [Google Scholar]
  43. Reid M. S., Bieleski R. L. A simple apparatus for vertical flat-sheet polyacrylamide gel electrophoresis. Anal Biochem. 1968 Mar;22(3):374–381. doi: 10.1016/0003-2697(68)90278-9. [DOI] [PubMed] [Google Scholar]
  44. Richards R. I., Wells J. R. Chicken globin genes. Nucleotide sequence of cDNA clones coding for the alpha-globin expressed during hemolytic anemia. J Biol Chem. 1980 Oct 10;255(19):9306–9311. [PubMed] [Google Scholar]
  45. Rovera G., Magarian C., Borun T. W. Resolution of hemoglobin subunits by electrophoresis in acid urea polyacrylamide gels containing Triton X-100. Anal Biochem. 1978 Apr;85(2):506–518. doi: 10.1016/0003-2697(78)90248-8. [DOI] [PubMed] [Google Scholar]
  46. Stalder J., Larsen A., Engel J. D., Dolan M., Groudine M., Weintraub H. Tissue-specific DNA cleavages in the globin chromatin domain introduced by DNAase I. Cell. 1980 Jun;20(2):451–460. doi: 10.1016/0092-8674(80)90631-5. [DOI] [PubMed] [Google Scholar]
  47. Steinberg R. A., O'Farrell P. H., Friedrich U., Coffino P. Mutations causing charge alterations in regulatory subunits of the cAMP-dependent protein kinase of cultured S49 lymphoma cells. Cell. 1977 Mar;10(3):381–391. doi: 10.1016/0092-8674(77)90025-3. [DOI] [PubMed] [Google Scholar]
  48. Sutter D., Doerfler W. Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression. Proc Natl Acad Sci U S A. 1980 Jan;77(1):253–256. doi: 10.1073/pnas.77.1.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Terada M., Epner E., Nudel U., Salmon J., Fibach E., Rifkind R. A., Marks P. A. Induction of murine erythroleukemia differentiation by actinomycin D. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2795–2799. doi: 10.1073/pnas.75.6.2795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weintraub H., Campbell G. le M., Holtzer H. Primitive erythropoiesis in early chick embryogenesis. I. Cell cycle kinetics and the control of cell division. J Cell Biol. 1971 Sep;50(3):652–668. doi: 10.1083/jcb.50.3.652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Weintraub H., Groudine M. Chromosomal subunits in active genes have an altered conformation. Science. 1976 Sep 3;193(4256):848–856. doi: 10.1126/science.948749. [DOI] [PubMed] [Google Scholar]
  52. Weisbrod S., Weintraub H. Isolation of actively transcribed nucleosomes using immobilized HMG 14 and 17 and an analysis of alpha-globin chromatin. Cell. 1981 Feb;23(2):391–400. doi: 10.1016/0092-8674(81)90134-3. [DOI] [PubMed] [Google Scholar]

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