Molecular cloning of human ornithine aminotransferase mRNA

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 approximately equal to 4 kDa, suggesting a precursor protein. lambda gt11 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 (lambda gtRB315) 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 seven 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 approximately equal to 2.2 kilobases. The level of OATase mRNA in the normal human retina is approximately equal to 1/100th the level of rhodopsin mRNA and 1/5th to 1/10th the level present in the retinoblastoma cells.