Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1989 Oct;84(4):1243–1252. doi: 10.1172/JCI114291

Sequence and exon-intron organization of the DNA encoding the alpha I domain of human spectrin. Application to the study of mutations causing hereditary elliptocytosis.

K E Sahr 1, T Tobe 1, A Scarpa 1, K Laughinghouse 1, S L Marchesi 1, P Agre 1, A J Linnenbach 1, V T Marchesi 1, B G Forget 1
PMCID: PMC329784  PMID: 2794061

Abstract

We have determined the exon-intron organization and the nucleotide sequence of the exons and their flanking intronic DNA in cloned genomic DNA that encodes the first 526 amino acids of the alpha I domain of the human red cell spectrin polypeptide chain. From the gene sequence we designed oligonucleotide primers to use in the polymerase chain reaction technique to amplify the appropriate exons in DNA from individuals with three variants of hereditary elliptocytosis characterized by the presence of abnormal alpha I spectrin peptides, 46-50 and 65-68 kD in size, in partial tryptic digests of spectrin. The alpha I/68-kD abnormality resulted from a duplication of leucine codon 148 in exon 4: TTG-CTG to TTG-TTG-CTG. The alpha I/50a defect was associated in different individuals with two separate single base changes in exon 6: CTG to CCG (leucine to proline) encoding residue 254, and TCC to CCC (serine to proline) encoding residue 255. In another individual with the alpha I/50a polypeptide defect, the nucleotide sequence encoding amino acid residues 221 through 264 was normal. The alpha I/50b abnormality resulted from a single base change of CAG (glutamine) to CCG (proline) encoding residue 465 in exon 11 in two unrelated individuals. In a third individual with alpha I/50b-kD hereditary elliptocytosis, the entire exon encoding residues 445 through 490 was normal. The relationship of the alpha I domain polypeptide structure to these mutations and the organization of the gene is discussed.

Full text

PDF
1243

Images in this article

1248Image
on p.1248
1249Image
on p.1249
1250Image
on p.1250

Selected References

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

  1. Alloisio N., Guetarni D., Morlé L., Pothier B., Ducluzeau M. T., Soun A., Colonna P., Clerc M., Philippe N., Delaunay J. Sp alpha I/65 hereditary elliptocytosis in North Africa. Am J Hematol. 1986 Oct;23(2):113–122. doi: 10.1002/ajh.2830230205. [DOI] [PubMed] [Google Scholar]
  2. Bennett V. The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu Rev Biochem. 1985;54:273–304. doi: 10.1146/annurev.bi.54.070185.001421. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Efstratiadis A., Posakony J. W., Maniatis T., Lawn R. M., O'Connell C., Spritz R. A., DeRiel J. K., Forget B. G., Weissman S. M., Slightom J. L. The structure and evolution of the human beta-globin gene family. Cell. 1980 Oct;21(3):653–668. doi: 10.1016/0092-8674(80)90429-8. [DOI] [PubMed] [Google Scholar]
  5. Garbarz M., Devaux I., Grandchamp B., Picat C., Dhermy D., Lecomte M. C., Boivin P., Sahr K. E., Forget B. Recherche de l'anomalie génétique dans une forme hémolytique d'elliptocytose héréditaire avec homozygotie pour le variant spectrine alpha I/74. C R Acad Sci III. 1989;308(2):43–48. [PubMed] [Google Scholar]
  6. Garbarz M., Lecomte M. C., Dhermy D., Feo C., Chaveroche I., Gautero H., Bournier O., Picat C., Goepp A., Boivin P. Double inheritance of an alpha I/65 spectrin variant in a child with homozygous elliptocytosis. Blood. 1986 Jun;67(6):1661–1667. [PubMed] [Google Scholar]
  7. Gyllensten U. B., Erlich H. A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652–7656. doi: 10.1073/pnas.85.20.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Knowles W. J., Morrow J. S., Speicher D. W., Zarkowsky H. S., Mohandas N., Mentzer W. C., Shohet S. B., Marchesi V. T. Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis. J Clin Invest. 1983 Jun;71(6):1867–1877. doi: 10.1172/JCI110942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Koenig M., Monaco A. P., Kunkel L. M. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell. 1988 Apr 22;53(2):219–228. doi: 10.1016/0092-8674(88)90383-2. [DOI] [PubMed] [Google Scholar]
  10. Lawler J., Coetzer T. L., Palek J., Jacob H. S., Luban N. Sp alpha I/65: a new variant of the alpha subunit of spectrin in hereditary elliptocytosis. Blood. 1985 Sep;66(3):706–709. [PubMed] [Google Scholar]
  11. Lawler J., Palek J., Liu S. C., Prchal J., Butler W. M. Molecular heterogeneity of hereditary pyropoikilocytosis: identification of a second variant of the spectrin alpha-subunit. Blood. 1983 Dec;62(6):1182–1189. [PubMed] [Google Scholar]
  12. 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]
  13. Lecomte M. C., Dhermy D., Gautero H., Bournier O., Galand C., Boivin P. L'elliptocytose héréditaire en Afrique de l'Ouest: fréquence et répartition des variants de la spectrine. C R Acad Sci III. 1988;306(2):43–46. [PubMed] [Google Scholar]
  14. Lecomte M. C., Dhermy D., Solis C., Ester A., Féo C., Gautero H., Bournier O., Boivin P. A new abnormal variant of spectrin in black patients with hereditary elliptocytosis. Blood. 1985 May;65(5):1208–1217. [PubMed] [Google Scholar]
  15. Lecomte M. C., Garbarz M., Grandchamp B., Féo C., Gautero H., Devaux I., Bournier O., Galand C., d'Auriol L., Galibert F. Sp alpha I/78: a mutation of the alpha I spectrin domain in a white kindred with HE and HPP phenotypes. Blood. 1989 Aug 15;74(3):1126–1133. [PubMed] [Google Scholar]
  16. Linnenbach A. J., Speicher D. W., Marchesi V. T., Forget B. G. Cloning of a portion of the chromosomal gene for human erythrocyte alpha-spectrin by using a synthetic gene fragment. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2397–2401. doi: 10.1073/pnas.83.8.2397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marchesi S. L., Knowles W. J., Morrow J. S., Bologna M., Marchesi V. T. Abnormal spectrin in hereditary elliptocytosis. Blood. 1986 Jan;67(1):141–151. [PubMed] [Google Scholar]
  18. Marchesi S. L., Letsinger J. T., Speicher D. W., Marchesi V. T., Agre P., Hyun B., Gulati G. Mutant forms of spectrin alpha-subunits in hereditary elliptocytosis. J Clin Invest. 1987 Jul;80(1):191–198. doi: 10.1172/JCI113047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marchesi V. T. Stabilizing infrastructure of cell membranes. Annu Rev Cell Biol. 1985;1:531–561. doi: 10.1146/annurev.cb.01.110185.002531. [DOI] [PubMed] [Google Scholar]
  20. Marotta C. A., Wilson J. T., Forget B. G., Weissman S. M. Human beta-globin messenger RNA. III. Nucleotide sequences derived from complementary DNA. J Biol Chem. 1977 Jul 25;252(14):5040–5053. [PubMed] [Google Scholar]
  21. Morlé L., Morlé F., Roux A. F., Godet J., Forget B. G., Denoroy L., Garbarz M., Dhermy D., Kastally R., Delaunay J. Spectrin Tunis (Sp alpha I/78), an elliptocytogenic variant, is due to the CGG----TGG codon change (Arg----Trp) at position 35 of the alpha I domain. Blood. 1989 Aug 1;74(2):828–832. [PubMed] [Google Scholar]
  22. Morrow J. S., Speicher D. W., Knowles W. J., Hsu C. J., Marchesi V. T. Identification of functional domains of human erythrocyte spectrin. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6592–6596. doi: 10.1073/pnas.77.11.6592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Palek J. Hereditary elliptocytosis, spherocytosis and related disorders: consequences of a deficiency or a mutation of membrane skeletal proteins. Blood Rev. 1987 Sep;1(3):147–168. doi: 10.1016/0268-960x(87)90031-2. [DOI] [PubMed] [Google Scholar]
  25. Roux A. F., Morlé F., Guetarni D., Colonna P., Sahr K., Forget B. G., Delaunay J., Godet J. Molecular basis of Sp alpha I/65 hereditary elliptocytosis in North Africa: insertion of a TTG triplet between codons 147 and 149 in the alpha-spectrin gene from five unrelated families. Blood. 1989 Jun;73(8):2196–2201. [PubMed] [Google Scholar]
  26. Saiki R. K., Bugawan T. L., Horn G. T., Mullis K. B., Erlich H. A. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature. 1986 Nov 13;324(6093):163–166. doi: 10.1038/324163a0. [DOI] [PubMed] [Google Scholar]
  27. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  30. Speicher D. W., Davis G., Marchesi V. T. Structure of human erythrocyte spectrin. II. The sequence of the alpha-I domain. J Biol Chem. 1983 Dec 25;258(24):14938–14947. [PubMed] [Google Scholar]
  31. Speicher D. W., Marchesi V. T. Erythrocyte spectrin is comprised of many homologous triple helical segments. Nature. 1984 Sep 13;311(5982):177–180. doi: 10.1038/311177a0. [DOI] [PubMed] [Google Scholar]
  32. Speicher D. W., Morrow J. S., Knowles W. J., Marchesi V. T. A structural model of human erythrocyte spectrin. Alignment of chemical and functional domains. J Biol Chem. 1982 Aug 10;257(15):9093–9101. [PubMed] [Google Scholar]
  33. Streisinger G., Okada Y., Emrich J., Newton J., Tsugita A., Terzaghi E., Inouye M. Frameshift mutations and the genetic code. This paper is dedicated to Professor Theodosius Dobzhansky on the occasion of his 66th birthday. Cold Spring Harb Symp Quant Biol. 1966;31:77–84. doi: 10.1101/sqb.1966.031.01.014. [DOI] [PubMed] [Google Scholar]
  34. Winkelmann J. C., Leto T. L., Watkins P. C., Eddy R., Shows T. B., Linnenbach A. J., Sahr K. E., Kathuria N., Marchesi V. T., Forget B. G. Molecular cloning of the cDNA for human erythrocyte beta-spectrin. Blood. 1988 Jul;72(1):328–334. [PubMed] [Google Scholar]
  35. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES