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. 1986 Dec;39(6):763–774.

Human acid beta-glucosidase: Northern blot and S1 nuclease analysis of mRNA from HeLa cells and normal and Gaucher disease fibroblasts.

P N Graves, G A Grabowski, M D Ludman, P Palese, F I Smith
PMCID: PMC1684132  PMID: 3026174

Abstract

Human acid beta-glucosidase (beta-Glc) mRNA was evaluated by dot blot, Northern blot, and S1 nuclease analyses of extracts of HeLa cells and cultured fibroblasts from normal individuals and Gaucher disease patients. Dot blot quantitation indicated an equal concentration of specific mRNA in all sources. Northern blot analyses demonstrated the presence of three poly (A)+ mRNAs of about 5,600, 2,500, and 2,000 nucleotides in length from all cell extracts. All three mRNAs were present in normal amounts in fibroblast extracts from several subtypes and variants of Gaucher disease. The largest poly (A)+ mRNA species was thought to represent an unspliced nuclear precursor for the two smaller beta-Glc mRNAs. S1 nuclease analyses, using SP6 transcripts of beta-Glc cDNA, indicated that the 2,000 nucleotide mRNA differs from the 2,500 nucleotide form at both the 5' and 3' ends. These results are consistent with the use of an alternate 5' splice site and 3' polyadenylation signal. These results also suggest that the subtype and variants of Gaucher disease result from single base alterations that lead to the synthesis of defective beta-Glc.

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Selected References

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

  1. Amara S. G., Evans R. M., Rosenfeld M. G. Calcitonin/calcitonin gene-related peptide transcription unit: tissue-specific expression involves selective use of alternative polyadenylation sites. Mol Cell Biol. 1984 Oct;4(10):2151–2160. doi: 10.1128/mcb.4.10.2151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amara S. G. Peptide diversity from exon choice: RNA processing regulation in the neuroendocrine system. Mol Cell Endocrinol. 1985 Oct;42(3):191–205. doi: 10.1016/0303-7207(85)90049-8. [DOI] [PubMed] [Google Scholar]
  3. Arnheim N. Characterization of mouse ribosomal gene fragments purified by molecular cloning. Gene. 1979 Oct;7(2):83–96. doi: 10.1016/0378-1119(79)90025-8. [DOI] [PubMed] [Google Scholar]
  4. BRINN L., GLABMAN S. Gaucher's disease without splenomegaly. Oldest patient on record, with review. N Y State J Med. 1962 Jul 15;62:2346–2354. [PubMed] [Google Scholar]
  5. Beutler E., Kuhl W., Sorge J. Cross-reacting material in Gaucher disease fibroblasts. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6506–6510. doi: 10.1073/pnas.81.20.6506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dodemont H. J., Soriano P., Quax W. J., Ramaekers F., Lenstra J. A., Groenen M. A., Bernardi G., Bloemendal H. The genes coding for the cytoskeletal proteins actin and vimentin in warm-blooded vertebrates. EMBO J. 1982;1(2):167–171. doi: 10.1002/j.1460-2075.1982.tb01142.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Franklin R. M. Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc Natl Acad Sci U S A. 1966 Jun;55(6):1504–1511. doi: 10.1073/pnas.55.6.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gonzalez I. L., Gorski J. L., Campen T. J., Dorney D. J., Erickson J. M., Sylvester J. E., Schmickel R. D. Variation among human 28S ribosomal RNA genes. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7666–7670. doi: 10.1073/pnas.82.22.7666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gonzalez I. L., Schmickel R. D. The human 18S ribosomal RNA gene: evolution and stability. Am J Hum Genet. 1986 Apr;38(4):419–427. [PMC free article] [PubMed] [Google Scholar]
  10. Grabowski G. A., Dinur T., Osiecki K. M., Kruse J. R., Legler G., Gatt S. Gaucher disease types 1, 2, and 3: differential mutations of the acid beta-glucosidase active site identified with conduritol B epoxide derivatives and sphingosine. Am J Hum Genet. 1985 May;37(3):499–510. [PMC free article] [PubMed] [Google Scholar]
  11. Grabowski G. A., Goldblatt J., Dinur T., Kruse J., Svennerholm L., Gatt S., Desnick R. J. Genetic heterogeneity in Gaucher disease: physicokinetic and immunologic studies of the residual enzyme in cultured fibroblasts from non-neuronopathic and neuronopathic patients. Am J Med Genet. 1985 Jul;21(3):529–549. doi: 10.1002/ajmg.1320210316. [DOI] [PubMed] [Google Scholar]
  12. Grabowski G. A., Osiecki-Newman K., Dinur T., Fabbro D., Legler G., Gatt S., Desnick R. J. Human acid beta-glucosidase. Use of conduritol B epoxide derivatives to investigate the catalytically active normal and Gaucher disease enzymes. J Biol Chem. 1986 Jun 25;261(18):8263–8269. [PubMed] [Google Scholar]
  13. Hodson P., Goldblatt J., Beighton P. Non-neuropathic Gaucher disease presenting in infancy. Arch Dis Child. 1979 Sep;54(9):707–709. doi: 10.1136/adc.54.9.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. MEDOFF A. S., BAYRD E. D. Gaucher's disease in 29 cases: hematologic complications and effect of splenectomy. Ann Intern Med. 1954 Mar;40(3):481–492. doi: 10.7326/0003-4819-40-3-481. [DOI] [PubMed] [Google Scholar]
  15. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Myerowitz R., Piekarz R., Neufeld E. F., Shows T. B., Suzuki K. Human beta-hexosaminidase alpha chain: coding sequence and homology with the beta chain. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7830–7834. doi: 10.1073/pnas.82.23.7830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pentchev P. G., Neumeyer B., Svennerholm L., Groth C. G., Brady R. O. Immunological and catalytic quantitation of splenic glucocerebrosidase from the three clinical forms of Gaucher disease. Am J Hum Genet. 1983 Jul;35(4):621–628. [PMC free article] [PubMed] [Google Scholar]
  19. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  20. Schenborn E. T., Mierendorf R. C., Jr A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res. 1985 Sep 11;13(17):6223–6236. doi: 10.1093/nar/13.17.6223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sheiness D., Darnell J. E. Polyadenylic acid segment in mRNA becomes shorter with age. Nat New Biol. 1973 Feb 28;241(113):265–268. doi: 10.1038/newbio241265a0. [DOI] [PubMed] [Google Scholar]
  22. Sorge J., West C., Westwood B., Beutler E. Molecular cloning and nucleotide sequence of human glucocerebrosidase cDNA. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7289–7293. doi: 10.1073/pnas.82.21.7289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tsuji S., Choudary P. V., Martin B. M., Winfield S., Barranger J. A., Ginns E. I. Nucleotide sequence of cDNA containing the complete coding sequence for human lysosomal glucocerebrosidase. J Biol Chem. 1986 Jan 5;261(1):50–53. [PubMed] [Google Scholar]
  24. Wickens M., Stephenson P. Role of the conserved AAUAAA sequence: four AAUAAA point mutants prevent messenger RNA 3' end formation. Science. 1984 Nov 30;226(4678):1045–1051. doi: 10.1126/science.6208611. [DOI] [PubMed] [Google Scholar]

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