Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA
- Mount Sinai School of Medicine, New York, NY (USA)
Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 {times} 10{sup 6} recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria.
- OSTI ID:
- 5478379
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America; (USA), Vol. 85:19; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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HEREDITARY DISEASES
BIOCHEMISTRY
HYDRO-LYASES
DNA-CLONING
RECOMBINANT DNA
DNA SEQUENCING
AMINO ACID SEQUENCE
BIOSYNTHESIS
HEME
LIQUID COLUMN CHROMATOGRAPHY
MAN
PHOSPHORUS 32
PORPHYRINS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBON-OXYGEN LYASES
CARBOXYLIC ACIDS
CHEMISTRY
CHROMATOGRAPHY
CLONING
DAYS LIVING RADIOISOTOPES
DISEASES
DNA
DNA HYBRIDIZATION
ENZYMES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYBRIDIZATION
ISOTOPES
LIGHT NUCLEI
LYASES
MAMMALS
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS ISOTOPES
PIGMENTS
PRIMATES
RADIOISOTOPES
SEPARATION PROCESSES
STRUCTURAL CHEMICAL ANALYSIS
SYNTHESIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques