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. 1983 Apr;80(8):2318–2322. doi: 10.1073/pnas.80.8.2318

"Silent" nucleotide substitution in a beta+-thalassemia globin gene activates splice site in coding sequence RNA.

M E Goldsmith, R K Humphries, T Ley, A Cline, J A Kantor, A W Nienhuis
PMCID: PMC393811  PMID: 6572978

Abstract

A beta+-thalassemia globin gene was isolated from the genome of a Black individual by molecular cloning. DNA sequence analysis revealed only a single difference between this gene and the normal human beta-globin gene--adenine is substituted for thymine in the third position of codon 24. Codon 24 in both the normal gene (GGT) and the beta+-thalassemia gene (GGA) encodes glycine. The function of this beta+-thalassemia gene was compared to the function of the normal human beta-globin gene in monkey kidney cells by using plasmid expression vectors. The codon 24 substitution activates a 5' splice site that involves the guanine-thymine dinucleotide present in codon 25, 16 nucleotides upstream from the normal exon 1-intron I boundary. The splice, involving the abnormal 5' site in codon 25, is completed with the normal 3' splice site at the end of intron I. This splicing abnormality leads to a 75% decrease in the accumulation of normally processed beta-globin mRNA, thereby causing the beta+-thalassemia phenotype.

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Selected References

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

  1. Antonarakis S. E., Boehm C. D., Giardina P. J., Kazazian H. H., Jr Nonrandom association of polymorphic restriction sites in the beta-globin gene cluster. Proc Natl Acad Sci U S A. 1982 Jan;79(1):137–141. doi: 10.1073/pnas.79.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banerji J., Rusconi S., Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell. 1981 Dec;27(2 Pt 1):299–308. doi: 10.1016/0092-8674(81)90413-x. [DOI] [PubMed] [Google Scholar]
  3. Benz E. J., Jr, Scarpa A. L., Tonkonow B. L., Pearson H. A., Ritchey A. K. Posttranscriptional defects in beta-globin messenger RNA metabolism in beta-thalassemia: abnormal accumulation of beta-messenger RNA precursor sequences. J Clin Invest. 1981 Dec;68(6):1529–1538. doi: 10.1172/JCI110407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  5. Busslinger M., Moschonas N., Flavell R. A. Beta + thalassemia: aberrant splicing results from a single point mutation in an intron. Cell. 1981 Dec;27(2 Pt 1):289–298. doi: 10.1016/0092-8674(81)90412-8. [DOI] [PubMed] [Google Scholar]
  6. Chang J. C., Kan Y. W. beta 0 thalassemia, a nonsense mutation in man. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2886–2889. doi: 10.1073/pnas.76.6.2886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Felber B. K., Orkin S. H., Hamer D. H. Abnormal RNA splicing causes one form of alpha thalassemia. Cell. 1982 Jul;29(3):895–902. doi: 10.1016/0092-8674(82)90451-2. [DOI] [PubMed] [Google Scholar]
  8. Flavell R. A., Bernards R., Kooter J. M., de Boer E., Little P. F., Annison G., Williamson R. The structure of the human beta-globin gene in beta-thalassaemia. Nucleic Acids Res. 1979 Jun 25;6(8):2749–2760. doi: 10.1093/nar/6.8.2749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fukumaki Y., Ghosh P. K., Benz E. J., Jr, Reddy V. B., Lebowitz P., Forget B. G., Weissman S. M. Abnormally spliced messenger RNA in erythroid cells from patients with beta+ thalassemia and monkey cells expressing a cloned beta+-thalassemic gene. Cell. 1982 Mar;28(3):585–593. doi: 10.1016/0092-8674(82)90213-6. [DOI] [PubMed] [Google Scholar]
  10. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  11. Gorski J., Fiori M., Mach B. A new nonsense mutation as the molecular basis for beta thalassaemia. J Mol Biol. 1982 Jan 25;154(3):537–540. doi: 10.1016/s0022-2836(82)80012-0. [DOI] [PubMed] [Google Scholar]
  12. Humphries R. K., Ley T., Turner P., Moulton A. D., Nienhuis A. W. Differences in human alpha-, beta- and delta-globin gene expression in monkey kidney cells. Cell. 1982 Aug;30(1):173–183. doi: 10.1016/0092-8674(82)90023-x. [DOI] [PubMed] [Google Scholar]
  13. Jeffreys A. J. DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man. Cell. 1979 Sep;18(1):1–10. doi: 10.1016/0092-8674(79)90348-9. [DOI] [PubMed] [Google Scholar]
  14. Kan Y. W., Lee K. Y., Furbetta M., Angius A., Cao A. Polymorphism of DNA sequence in the beta-globin gene region. Application to prenatal diagnosis of beta 0 thalassemia in Sardinia. N Engl J Med. 1980 Jan 24;302(4):185–188. doi: 10.1056/NEJM198001243020401. [DOI] [PubMed] [Google Scholar]
  15. Kantor J. A., Turner P. H., Nienhuis A. W. Beta Thalassemia: mutations which affect processing of the beta-Globin mRNA precursor. Cell. 1980 Aug;21(1):149–157. doi: 10.1016/0092-8674(80)90122-1. [DOI] [PubMed] [Google Scholar]
  16. Kretschmer P. J., Coon H. C., Davis A., Harrison M., Nienhuis A. W. Hemoglobin switching in sheep. Isolation of the fetal gamma-globin gene and demonstration that the fetal gamma- and adult beta A-globin genes lie within eight kilobase segments of homologous DNA. J Biol Chem. 1981 Feb 25;256(4):1975–1982. [PubMed] [Google Scholar]
  17. Kretschmer P. J., Kaufman R. E., Coon H. C., Chen M. J., Geist C. E., Nienhuis A. W. Hemoglobin switching in sheep. Cloning and characterization of the beta A and beta-like embryonic globin genes from genomic DNA. J Biol Chem. 1980 Apr 10;255(7):3204–3211. [PubMed] [Google Scholar]
  18. Lawn R. M., Efstratiadis A., O'Connell C., Maniatis T. The nucleotide sequence of the human beta-globin gene. Cell. 1980 Oct;21(3):647–651. doi: 10.1016/0092-8674(80)90428-6. [DOI] [PubMed] [Google Scholar]
  19. Lerner M. R., Boyle J. A., Mount S. M., Wolin S. L., Steitz J. A. Are snRNPs involved in splicing? Nature. 1980 Jan 10;283(5743):220–224. doi: 10.1038/283220a0. [DOI] [PubMed] [Google Scholar]
  20. Ley T. J., Anagnou N. P., Pepe G., Nienhuis A. W. RNA processing errors in patients with beta-thalassemia. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4775–4779. doi: 10.1073/pnas.79.15.4775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  22. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  23. Maquat L. E., Kinniburgh A. J., Beach L. R., Honig G. R., Lazerson J., Ershler W. B., Ross J. Processing of human beta-globin mRNA precursor to mRNA is defective in three patients with beta+-thalassemia. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4287–4291. doi: 10.1073/pnas.77.7.4287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maquat L. E., Kinniburgh A. J., Rachmilewitz E. A., Ross J. Unstable beta-globin mRNA in mRNA-deficient beta o thalassemia. Cell. 1981 Dec;27(3 Pt 2):543–553. doi: 10.1016/0092-8674(81)90396-2. [DOI] [PubMed] [Google Scholar]
  25. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mellon P., Parker V., Gluzman Y., Maniatis T. Identification of DNA sequences required for transcription of the human alpha 1-globin gene in a new SV40 host-vector system. Cell. 1981 Dec;27(2 Pt 1):279–288. doi: 10.1016/0092-8674(81)90411-6. [DOI] [PubMed] [Google Scholar]
  27. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moschonas N., de Boer E., Grosveld F. G., Dahl H. H., Wright S., Shewmaker C. K., Flavell R. A. Structure and expression of a cloned beta o thalassaemic globin gene. Nucleic Acids Res. 1981 Sep 11;9(17):4391–4401. doi: 10.1093/nar/9.17.4391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Myers R. M., Tjian R. Construction and analysis of simian virus 40 origins defective in tumor antigen binding and DNA replication. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6491–6495. doi: 10.1073/pnas.77.11.6491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nienhuis A. W., Turner P., Benz E. J., Jr Relative stability of alpha- and beta-globin messenger RNAs in homozygous beta+ thalassemia. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3960–3964. doi: 10.1073/pnas.74.9.3960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Orkin S. H., Goff S. C. Nonsense and frameshift mutations in beta 0-thalassemia detected in cloned beta-globin genes. J Biol Chem. 1981 Oct 10;256(19):9782–9784. [PubMed] [Google Scholar]
  33. Orkin S. H., Kazazian H. H., Jr, Antonarakis S. E., Goff S. C., Boehm C. D., Sexton J. P., Waber P. G., Giardina P. J. Linkage of beta-thalassaemia mutations and beta-globin gene polymorphisms with DNA polymorphisms in human beta-globin gene cluster. Nature. 1982 Apr 15;296(5858):627–631. doi: 10.1038/296627a0. [DOI] [PubMed] [Google Scholar]
  34. Orkin S. H., Kazazian H. H., Jr, Antonarakis S. E., Ostrer H., Goff S. C., Sexton J. P. Abnormal RNA processing due to the exon mutation of beta E-globin gene. Nature. 1982 Dec 23;300(5894):768–769. doi: 10.1038/300768a0. [DOI] [PubMed] [Google Scholar]
  35. Orkin S. H., Old J. M., Weatherall D. J., Nathan D. G. Partial deletion of beta-globin gene DNA in certain patients with beta 0-thalassemia. Proc Natl Acad Sci U S A. 1979 May;76(5):2400–2404. doi: 10.1073/pnas.76.5.2400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Poncz M., Ballantine M., Solowiejczyk D., Barak I., Schwartz E., Surrey S. beta-Thalassemia in a Kurdish Jew. Single base changes in the T-A-T-A box. J Biol Chem. 1982 Jun 10;257(11):5994–5996. [PubMed] [Google Scholar]
  37. Rogers J., Wall R. A mechanism for RNA splicing. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1877–1879. doi: 10.1073/pnas.77.4.1877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spence S. E., Pergolizzi R. G., Donovan-Peluso M., Kosche K. A., Dobkin C. S., Bank A. Five nucleotide changes in the large intervening sequence of a beta globin gene in a beta+ thalassemia patient. Nucleic Acids Res. 1982 Feb 25;10(4):1283–1294. doi: 10.1093/nar/10.4.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Spritz R. A., Jagadeeswaran P., Choudary P. V., Biro P. A., Elder J. T., deRiel J. K., Manley J. L., Gefter M. L., Forget B. G., Weissman S. M. Base substitution in an intervening sequence of a beta+-thalassemic human globin gene. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2455–2459. doi: 10.1073/pnas.78.4.2455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Trecartin R. F., Liebhaber S. A., Chang J. C., Lee K. Y., Kan Y. W., Furbetta M., Angius A., Cao A. beta zero thalassemia in Sardinia is caused by a nonsense mutation. J Clin Invest. 1981 Oct;68(4):1012–1017. doi: 10.1172/JCI110323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Treisman R., Proudfoot N. J., Shander M., Maniatis T. A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing. Cell. 1982 Jul;29(3):903–911. doi: 10.1016/0092-8674(82)90452-4. [DOI] [PubMed] [Google Scholar]
  43. Tuan D., Biro P. A., deRiel J. K., Lazarus H., Forget B. G. Restriction endonuclease mapping of the human gamma globin gene loci. Nucleic Acids Res. 1979 Jun 11;6(7):2519–2544. doi: 10.1093/nar/6.7.2519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weatherall D. J., Clegg J. B. Thalassemia revisited. Cell. 1982 May;29(1):7–9. doi: 10.1016/0092-8674(82)90084-8. [DOI] [PubMed] [Google Scholar]
  45. Weaver R. F., Weissmann C. Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini of 15 S beta-globin mRNA precursor and mature 10 s beta-globin mRNA have identical map coordinates. Nucleic Acids Res. 1979 Nov 10;7(5):1175–1193. doi: 10.1093/nar/7.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Westaway D., Williamson R. An intron nucleotide sequence variant in a cloned beta +-thalassaemia globin gene. Nucleic Acids Res. 1981 Apr 24;9(8):1777–1788. doi: 10.1093/nar/9.8.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]

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