Structure and Expression of a Complementary DNA for the Nuclear Coded Precursor of Human Mitochondrial Ornithine Transcarbamylase

Abstract

Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria. We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA. The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues. Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues. The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli. The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC. Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.

Synthesis and Assembly of Mitochondrial Proteins

“…Proteins: (1) 70 kDa protein (yeast), Hase et al (1983); (2) cytochrome c peroxidase (yeast), Kaput et al (1982), Reid et al (1982); (3) cytochrome b 2 (yeast), Guiard (1985), Gasser et al (1982b); (4) Rieske Fe/S of bc, complex (N. crassa), Harnisch et al (1985), Hartl et al (1986); (5) cytochrome d (yeast), Sadler et al (1984), Gasser et al (1982b); (6) cytochrome oxidase V (yeast), Koerner et aJ. (1985); (7) cytochrome oxidase V (N. crassa), Sachs et al (1986); (8) cytochrome oxidase IV (bovine), Lomax et al (1984); (9) cytochrome P-450 (SCC) (bovine), Morohashi et al (1984); (10) cytochrome oxidase VI (yeast), Wright et al (1984) ; (11) ATPase IX (N. crassa), Viebrock et al (1982), Schmidt et al (1984); (12) ATPase IX-Pl (bovine), Gay and Walker (1985); (13) ATPase IX-P2 (bovine), Gay and Walker (1985); (14) cytochrome oxidase IV (yeast), Maarse et al (1984); (15) citrate synthase (yeast), Suissa et al (1984); (16) Ornithine aminotransferase (rat), Mueckler and Pitot (1985), Simmaco et al (1986); (17) aspartate aminotransferase (chicken), Jaussi et al (1985) ; (18) aspartate aminotransferase (porcine), Joh et al (1985); (19) Mn-superoxide dismutase (yeast), Marres et al (1985); (20) ATPase F, 5 subunit (N. crassa), Kruse and Sebald (1984); (21) 5-aminolevulinate synthase (yeast), Keng et al (1986), Urban-Grimal et al (1986); (22) alcohol dehydrogenase iso-III (yeast), Young and Pilgrim (1985); (23) Ornithine carbamoyltransferase (human), Horwich et al (1984); (24) Ornithine carbamoyltransferase (rat), Mclntyre et al (1...…”

Mutant alleles associated with late-onset ornithine transcarbamylase deficiency in male patients have recurrently arisen and have been retained in some populations

“…1 The gene spans 74 kb with an open reading frame of 1062 nucleotides distributed into 10 exons and 9 introns. 2,3 The phenotypes of females heterozygous for a mutant OTC allele vary from asymptomatic carrier state to overt, even fatal disease, depending first on the nature of the gene mutation, second on X-inactivation pattern and third on other genes and environmental factors. In contrast, in hemizygous male patients, the phenotype is determined by the nature of mutation and other yet unknown factors (other genes/environment).…”

Novel intragenic deletions and point mutations of the ornithine transcarbamylase gene in congenital hyperammonemia

“…Heterozygous females occasionally display symptoms ranging in severity from a mere dislike for protein-containing foods to recurrent episodes of acute hyperammonemia and lifethreatening comas (Rowe et al, 1986). The human OTC gene contains 10 exons encoding a 354-amino acid protein and maps to chromosome Xp21.1 (Horwich et al, 1984;Lindgren et al 1984;Hata et al, 1988). To date, more than 80 different point mutations and deletions or duplications have been reported in OTC deficiency (Tuchman, 1993;Tuchman and Plante, 1995;GilbertDussardier et al, 1996).…”