Abstract
L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency.
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- Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
- Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
- Fenton W. A., Hack A. M., Kraus J. P., Rosenberg L. E. Immunochemical studies of fibroblasts from patients with methylmalonyl-CoA mutase apoenzyme deficiency: detection of a mutation interfering with mitochondrial import. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1421–1424. doi: 10.1073/pnas.84.5.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fenton W. A., Hack A. M., Willard H. F., Gertler A., Rosenberg L. E. Purification and properties of methylmalonyl coenzyme A mutase from human liver. Arch Biochem Biophys. 1982 Apr 1;214(2):815–823. doi: 10.1016/0003-9861(82)90088-1. [DOI] [PubMed] [Google Scholar]
- Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- Grenett H. E., Ledley F. D., Reed L. L., Woo S. L. Full-length cDNA for rabbit tryptophan hydroxylase: functional domains and evolution of aromatic amino acid hydroxylases. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5530–5534. doi: 10.1073/pnas.84.16.5530. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henthorn P. S., Knoll B. J., Raducha M., Rothblum K. N., Slaughter C., Weiss M., Lafferty M. A., Fischer T., Harris H. Products of two common alleles at the locus for human placental alkaline phosphatase differ by seven amino acids. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5597–5601. doi: 10.1073/pnas.83.15.5597. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaufman R. J. Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors. Proc Natl Acad Sci U S A. 1985 Feb;82(3):689–693. doi: 10.1073/pnas.82.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kolhouse J. F., Utley C., Allen R. H. Isolation and characterization of methylmalonyl-CoA mutase from human placenta. J Biol Chem. 1980 Apr 10;255(7):2708–2712. [PubMed] [Google Scholar]
- Kolhouse J. F., Utley C., Fenton W. A., Rosenberg L. E. Immunochemical studies on cultured fibroblasts from patients with inherited methylmalonic acidemia. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7737–7741. doi: 10.1073/pnas.78.12.7737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kwok S. C., Ledley F. D., DiLella A. G., Robson K. J., Woo S. L. Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase. Biochemistry. 1985 Jan 29;24(3):556–561. doi: 10.1021/bi00324a002. [DOI] [PubMed] [Google Scholar]
- Ledley F. D., Grenett H. E., DiLella A. G., Kwok S. C., Woo S. L. Gene transfer and expression of human phenylalanine hydroxylase. Science. 1985 Apr 5;228(4695):77–79. doi: 10.1126/science.3856322. [DOI] [PubMed] [Google Scholar]
- Ledley F. D., Levy H. L., Shih V. E., Benjamin R., Mahoney M. J. Benign methylmalonic aciduria. N Engl J Med. 1984 Oct 18;311(16):1015–1018. doi: 10.1056/NEJM198410183111604. [DOI] [PubMed] [Google Scholar]
- Ledley F. D. Somatic gene therapy for human disease: background and prospects. Part I. J Pediatr. 1987 Jan;110(1):1–8. doi: 10.1016/s0022-3476(87)80278-0. [DOI] [PubMed] [Google Scholar]
- Ledley F. D. Somatic gene therapy for human disease: background and prospects. Part II. J Pediatr. 1987 Feb;110(2):167–174. doi: 10.1016/s0022-3476(87)80148-8. [DOI] [PubMed] [Google Scholar]
- Lockyer J., Cook R. G., Milstien S., Kaufman S., Woo S. L., Ledley F. D. Structure and expression of human dihydropteridine reductase. Proc Natl Acad Sci U S A. 1987 May;84(10):3329–3333. doi: 10.1073/pnas.84.10.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marvit J., DiLella A. G., Brayton K., Ledley F. D., Robson K. J., Woo S. L. GT to AT transition at a splice donor site causes skipping of the preceding exon in phenylketonuria. Nucleic Acids Res. 1987 Jul 24;15(14):5613–5628. doi: 10.1093/nar/15.14.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsui S. M., Mahoney M. J., Rosenberg L. E. The natural history of the inherited methylmalonic acidemias. N Engl J Med. 1983 Apr 14;308(15):857–861. doi: 10.1056/NEJM198304143081501. [DOI] [PubMed] [Google Scholar]
- 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]
- Rave N., Crkvenjakov R., Boedtker H. Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucleic Acids Res. 1979 Aug 10;6(11):3559–3567. doi: 10.1093/nar/6.11.3559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robson K. J., Chandra T., MacGillivray R. T., Woo S. L. Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4701–4705. doi: 10.1073/pnas.79.15.4701. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weinberger C., Hollenberg S. M., Ong E. S., Harmon J. M., Brower S. T., Cidlowski J., Thompson E. B., Rosenfeld M. G., Evans R. M. Identification of human glucocorticoid receptor complementary DNA clones by epitope selection. Science. 1985 May 10;228(4700):740–742. doi: 10.1126/science.2581314. [DOI] [PubMed] [Google Scholar]
- Willard H. F., Rosenberg L. E. Inherited methylmalonyl CoA mutase apoenzyme deficiency in human fibroblasts: evidence for allelic heterogeneity, genetic compounds, and codominant expression. J Clin Invest. 1980 Mar;65(3):690–698. doi: 10.1172/JCI109715. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wong G. G., Witek J. S., Temple P. A., Wilkens K. M., Leary A. C., Luxenberg D. P., Jones S. S., Brown E. L., Kay R. M., Orr E. C. Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science. 1985 May 17;228(4701):810–815. doi: 10.1126/science.3923623. [DOI] [PubMed] [Google Scholar]
- 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]