Molecular cloning of a cDNA for human pyruvate carboxylase. Structural relationship to other biotin-containing carboxylases and regulation of mRNA content in differentiating preadipocytes.

S O Freytag,K J Collier

doi:10.1016/s0021-9258(18)90822-7

Abstract

An oligonucleotide probe specific for the amino acid sequence at the biotin site in pyruvate carboxylase was used to screen a human liver cDNA library. Nine cDNA clones were isolated and three proved to be pyruvate carboxylase clones based on nucleotide sequencing and Northern blotting. The biotin site amino acid sequence of human pyruvate carboxylase agreed perfectly with that of the sheep enzyme in 14 consecutive positions. The highly conserved amino acid sequence, Ala-Met-Lys-Met, found at the biotin site in most biotin-containing carboxylases was also present in human pyruvate carboxylase. The termination codon was located 35 residues 3' to the lysine residue at which the biotin is attached. Therefore, the biotin cofactor is covalently linked near the carboxyl-terminal end of the carboxylase protein. These data are consistent with that observed for other biotin-containing carboxylases and strongly suggests that the genes encoding the biotin-containing carboxylases may have evolved from a common ancestral gene. Northern blotting of mRNA isolated from human, baboon, and rat liver demonstrated that the pyruvate carboxylase mRNA was 4.2 kilobase pairs in length in all species examined. Southern blot analysis of genomic DNA isolated from human-Chinese hamster somatic cell hybrids localized the pyruvate carboxylase gene on the long arm of human chromosome 11. The human cDNA was also used to quantitate pyruvate carboxylase mRNA levels in a differentiating mouse preadipocyte cell line. These data demonstrated that pyruvate carboxylase mRNA content increased 23-fold in 7 days after the onset of differentiation.

Highlights

EXPERIMENTAL PROCEDURES(Walter, 1976) and catalyzes the formation of oxalacetate from pyruvate and HCO, and utilizes ATP (Utter anKd eech, 1960)
From the $Department of Pediatrics, Section of Geneties, Baylor College of Medicine, Houston, Texas77030 and the ?Roche Research Center, Hoffmann La-RocheZnc., Nutley, New Jersey 07110
Nine cDNA synthesis of ofacetyl groups and reducing groups for transport clones were isolated and three proved to be pyruvate from the mitochondria to thecytosol; and othertissues where carboxylase clones based on nucleotide sequencing and it has an anapleurotic role in the formation of oxaloacetate

Summary

EXPERIMENTAL PROCEDURES

(Walter, 1976) and catalyzes the formation of oxalacetate from pyruvate and HCO, and utilizes ATP (Utter anKd eech, 1960). Hybridization was carried out in a solution of the same formulation but in addition containing 6 X lo6cpm/ml (5 X lo6cpm/filter) of the 32P-labeled14-. Putative positives were colony purified using the exact same X SSC, 5 X Denhardt's, 0.25 mg/ml of denatured salmon sperm DNA hybridization and washing conditions. Hybridization was carried out in a solution of the Subcloning of Biotin Site Sequence-Plasmid DNA was prepared same formulation but in addition containing 10% (w/v) dextran (Birnboim and Doly,1979) and digested with AluI and HaeIII to sulfate and 2 X IO6 cpm/lane of 32P-labeledphPC1. Coli strain JM 83 cells The filter was blot dried and radioactive bands were visualized by and bacteria containing a recombinant plasmid were selected by autoradiography using a Dupont Cronex Lightning Intensifying growth on agar plates containingampicillin (100pg/ml) and 5-bromo4-chloro-3-indolyl-~-~-galactopyranos(i1demg/plate).

RESULTS

Differentiated

Findings

DISCUSSION