Molecular cloning of human ornithine aminotransferase mRNA

George Inana, S. Totsuka, M. Redmond, T. Dougherty, J. Nagle, T. Shiono, T. Ohura, E. Kominami, N. Katunuma

Research output: Contribution to journalArticle

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Abstract

The isolation and characterization of a cDNA clone for the mRNA of human ornithine aminotransferase (OATase; ornithine-oxo-acid aminotransferase; L-ornithine:2-oxo-acid aminotransferase, EC 2.6.1.13), a nonabundant mitochondrial matrix enzyme that is severely deficient in a hereditary chorioretinal degenerative disease (gyrate atrophy), is described. Human liver, retina, and retinoblastoma (Y79) mRNAs were prepared and tested for the OATase mRNA content by in vitro translation, immunoprecipitation, and NaDodSO4/PAGE. The retinoblastoma cells were found to be expressing this enzyme at a relatively high level. The primary translation product of the OATase mRNA is larger than the pure OATase protein on NaDodSO4/PAGE by ≃4 kDa, suggesting a precursor protein. λgt11 cDNA libraries were prepared from the human mRNAs, and the recombinant clones were immunoscreened as plaques with two different preparations of rabbit anti-human OATase antibodies. A clone (λgtRB315) was isolated from the retinoblastoma library that reacts with both of the antibody preparations, and the DNA sequence of its 2.1-kilobase-pair cDNA insert was obtained. An open reading frame consisting of 1371 nucleotides is present in the sequence, and a putative translational initiation methionine codon is identified at position 55. A putative leader sequence consisting of 32 amino acid residues is identified, resulting in a precursor protein of 439 amino acid residues and a molecular mass of 48,534 Da and a mature protein of 407 residues and 45,136 Da. The amino acid sequences of 7 tryptic peptides (115 amino acid residues) of the pure human OATase were obtained by microsequencing. When the tryptic peptide and cDNA-derived amino acid sequences were compared, homologies in 111 of 115 residues, including a match of 20 consecutive residues, were observed. An RNA blot hybridization of 32P-labeled OATase cDNA to normal human retina and retinoblastoma mRNAs demonstrated an OATase mRNA species of ≃2.2 kilobases. The level of OATase mRNA in the normal human retina is ≃1/100th the level of rhodopsin mRNA and 1/5th to 1/10th the level present in the retinoblastoma cells.

Original languageEnglish
Pages (from-to)1203-1207
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume83
Issue number5
StatePublished - Nov 19 1986
Externally publishedYes

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Ornithine-Oxo-Acid Transaminase
Molecular Cloning
Retinoblastoma
Messenger RNA
Complementary DNA
Retina
Protein Precursors
Clone Cells
Amino Acids
Amino Acid Sequence
Gyrate Atrophy
Keto Acids
Peptides
Ornithine
Rhodopsin
Initiator Codon
Antibodies
Enzymes
Transaminases
Gene Library

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Inana, G., Totsuka, S., Redmond, M., Dougherty, T., Nagle, J., Shiono, T., ... Katunuma, N. (1986). Molecular cloning of human ornithine aminotransferase mRNA. Proceedings of the National Academy of Sciences of the United States of America, 83(5), 1203-1207.

Molecular cloning of human ornithine aminotransferase mRNA. / Inana, George; Totsuka, S.; Redmond, M.; Dougherty, T.; Nagle, J.; Shiono, T.; Ohura, T.; Kominami, E.; Katunuma, N.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 83, No. 5, 19.11.1986, p. 1203-1207.

Research output: Contribution to journalArticle

Inana, G, Totsuka, S, Redmond, M, Dougherty, T, Nagle, J, Shiono, T, Ohura, T, Kominami, E & Katunuma, N 1986, 'Molecular cloning of human ornithine aminotransferase mRNA', Proceedings of the National Academy of Sciences of the United States of America, vol. 83, no. 5, pp. 1203-1207.
Inana, George ; Totsuka, S. ; Redmond, M. ; Dougherty, T. ; Nagle, J. ; Shiono, T. ; Ohura, T. ; Kominami, E. ; Katunuma, N. / Molecular cloning of human ornithine aminotransferase mRNA. In: Proceedings of the National Academy of Sciences of the United States of America. 1986 ; Vol. 83, No. 5. pp. 1203-1207.
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AU - Totsuka, S.

AU - Redmond, M.

AU - Dougherty, T.

AU - Nagle, J.

AU - Shiono, T.

AU - Ohura, T.

AU - Kominami, E.

AU - Katunuma, N.

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N2 - The isolation and characterization of a cDNA clone for the mRNA of human ornithine aminotransferase (OATase; ornithine-oxo-acid aminotransferase; L-ornithine:2-oxo-acid aminotransferase, EC 2.6.1.13), a nonabundant mitochondrial matrix enzyme that is severely deficient in a hereditary chorioretinal degenerative disease (gyrate atrophy), is described. Human liver, retina, and retinoblastoma (Y79) mRNAs were prepared and tested for the OATase mRNA content by in vitro translation, immunoprecipitation, and NaDodSO4/PAGE. The retinoblastoma cells were found to be expressing this enzyme at a relatively high level. The primary translation product of the OATase mRNA is larger than the pure OATase protein on NaDodSO4/PAGE by ≃4 kDa, suggesting a precursor protein. λgt11 cDNA libraries were prepared from the human mRNAs, and the recombinant clones were immunoscreened as plaques with two different preparations of rabbit anti-human OATase antibodies. A clone (λgtRB315) was isolated from the retinoblastoma library that reacts with both of the antibody preparations, and the DNA sequence of its 2.1-kilobase-pair cDNA insert was obtained. An open reading frame consisting of 1371 nucleotides is present in the sequence, and a putative translational initiation methionine codon is identified at position 55. A putative leader sequence consisting of 32 amino acid residues is identified, resulting in a precursor protein of 439 amino acid residues and a molecular mass of 48,534 Da and a mature protein of 407 residues and 45,136 Da. The amino acid sequences of 7 tryptic peptides (115 amino acid residues) of the pure human OATase were obtained by microsequencing. When the tryptic peptide and cDNA-derived amino acid sequences were compared, homologies in 111 of 115 residues, including a match of 20 consecutive residues, were observed. An RNA blot hybridization of 32P-labeled OATase cDNA to normal human retina and retinoblastoma mRNAs demonstrated an OATase mRNA species of ≃2.2 kilobases. The level of OATase mRNA in the normal human retina is ≃1/100th the level of rhodopsin mRNA and 1/5th to 1/10th the level present in the retinoblastoma cells.

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