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. 1985 Feb 11;13(3):777–790. doi: 10.1093/nar/13.3.777

Beta-thalassemia resulting from a single nucleotide substitution in an acceptor splice site.

G F Atweh, N P Anagnou, J Shearin, B G Forget, R E Kaufman
PMCID: PMC341034  PMID: 2987809

Abstract

Beta-globin gene mutations which alter normal globin RNA splicing have confirmed the necessity of invariant nucleotides GT at donor splice sites. Functional consequences of point mutations in the invariant AG acceptor splice site have not been determined. We have isolated and characterized a beta-globin gene from a Black patient with beta-thalassemia intermedia which has an A-G transition at the usual intervening sequence 2 (IVS2) acceptor splice site. Functional analysis of transcripts produced by this mutant gene in a transient expression vector indicates that the mutation inactivates the normal acceptor splice site and results in some utilization of a cryptic splice site near position 580 of IVS2. This mutation would be expected to produce a beta-globin gene which results in no normal beta-globin mRNA.

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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., Irkin S. H., Cheng T. C., Scott A. F., Sexton J. P., Trusko S. P., Charache S., Kazazian H. H., Jr beta-Thalassemia in American Blacks: novel mutations in the "TATA" box and an acceptor splice site. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1154–1158. doi: 10.1073/pnas.81.4.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Antonarakis S. E., Orkin S. H., Kazazian H. H., Jr, Goff S. C., Boehm C. D., Waber P. G., Sexton J. P., Ostrer H., Fairbanks V. F., Chakravarti A. Evidence for multiple origins of the beta E-globin gene in Southeast Asia. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6608–6611. doi: 10.1073/pnas.79.21.6608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baird M., Driscoll C., Schreiner H., Sciarratta G. V., Sansone G., Niazi G., Ramirez F., Bank A. A nucleotide change at a splice junction in the human beta-globin gene is associated with beta 0-thalassemia. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4218–4221. doi: 10.1073/pnas.78.7.4218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Blattner F. R., Blechl A. E., Denniston-Thompson K., Faber H. E., Richards J. E., Slightom J. L., Tucker P. W., Smithies O. Cloning human fetal gamma globin and mouse alpha-type globin DNA: preparation and screening of shotgun collections. Science. 1978 Dec 22;202(4374):1279–1284. doi: 10.1126/science.725603. [DOI] [PubMed] [Google Scholar]
  7. Blin N., Stafford D. W. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 1976 Sep;3(9):2303–2308. doi: 10.1093/nar/3.9.2303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Breathnach R., Benoist C., O'Hare K., Gannon F., Chambon P. Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4853–4857. doi: 10.1073/pnas.75.10.4853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Chu G., Sharp P. A. SV40 DNA transfection of cells in suspension: analysis of efficiency of transcription and translation of T-antigen. Gene. 1981 Mar;13(2):197–202. doi: 10.1016/0378-1119(81)90008-1. [DOI] [PubMed] [Google Scholar]
  13. Dobkin C., Pergolizzi R. G., Bahre P., Bank A. Abnormal splice in a mutant human beta-globin gene not at the site of a mutation. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1184–1188. doi: 10.1073/pnas.80.5.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  18. Goldsmith M. E., Humphries R. K., Ley T., Cline A., Kantor J. A., Nienhuis A. W. "Silent" nucleotide substitution in a beta+-thalassemia globin gene activates splice site in coding sequence RNA. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2318–2322. doi: 10.1073/pnas.80.8.2318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heidecker G., Messing J., Gronenborn B. A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene. 1980 Jun;10(1):69–73. doi: 10.1016/0378-1119(80)90145-6. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Kimura A., Matsunaga E., Takihara Y., Nakamura T., Takagi Y., Lin S., Lee H. Structural analysis of a beta-thalassemia gene found in Taiwan. J Biol Chem. 1983 Mar 10;258(5):2748–2749. [PubMed] [Google Scholar]
  23. Kinniburgh A. J., Maquat L. E., Schedl T., Rachmilewitz E., Ross J. mRNA-deficient beta o-thalassemia results from a single nucleotide deletion. Nucleic Acids Res. 1982 Sep 25;10(18):5421–5427. doi: 10.1093/nar/10.18.5421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Lawn R. M., Fritsch E. F., Parker R. C., Blake G., Maniatis T. The isolation and characterization of linked delta- and beta-globin genes from a cloned library of human DNA. Cell. 1978 Dec;15(4):1157–1174. doi: 10.1016/0092-8674(78)90043-0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Ley T. J., Nienhuis A. W. A weak upstream promoter gives rise to long human beta-globin RNA molecules. Biochem Biophys Res Commun. 1983 May 16;112(3):1041–1048. doi: 10.1016/0006-291x(83)91723-0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. Orkin S. H., Kolodner R., Michelson A., Husson R. Cloning and direct examination of a structurally abnormal human beta 0-thalassemia globin gene. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3558–3562. doi: 10.1073/pnas.77.6.3558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Orkin S. H., Sexton J. P., Cheng T. C., Goff S. C., Giardina P. J., Lee J. I., Kazazian H. H., Jr ATA box transcription mutation in beta-thalassemia. Nucleic Acids Res. 1983 Jul 25;11(14):4727–4734. doi: 10.1093/nar/11.14.4727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Orkin S. H., Sexton J. P., Goff S. C., Kazazian H. H., Jr Inactivation of an acceptor RNA splice site by a short deletion in beta-thalassemia. J Biol Chem. 1983 Jun 25;258(12):7249–7251. [PubMed] [Google Scholar]
  41. 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]
  42. Rimm D. L., Horness D., Kucera J., Blattner F. R. Construction of coliphage lambda Charon vectors with BamHI cloning sites. Gene. 1980 Dec;12(3-4):301–309. doi: 10.1016/0378-1119(80)90113-4. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Treisman R., Orkin S. H., Maniatis T. Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes. Nature. 1983 Apr 14;302(5909):591–596. doi: 10.1038/302591a0. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Wieringa B., Meyer F., Reiser J., Weissmann C. Unusual splice sites revealed by mutagenic inactivation of an authentic splice site of the rabbit beta-globin gene. Nature. 1983 Jan 6;301(5895):38–43. doi: 10.1038/301038a0. [DOI] [PubMed] [Google Scholar]

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