Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Abstract

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.Asparagine synthetase (AS) is a housekeeping enzyme responsible for the biosynthesis of asparagine from glutamine and aspartic acid. The regulation of AS has been studied in cultured mammalian cells (for a review, see reference 1), and overproducing cell lines, some with amplified copies of the gene, have been isolated in response to drug selection (2, 3, 6, 11). Most mammalian cells express AS and regulate the level of activity in response to the concentration of asparagine in the medium and the extent of tRNA aminoacylation (4, 7). In contrast to normal cells, some tumor cells exhibit little or no AS activity and rely on the surrounding medium as a source of exogenous asparagine. This property has been exploited in the treatment of certain cancers, since the tumor cells can be selectively killed by the chemotherapeutic drug asparaginase which hydrolyzes circulating asparagine (9, 24). One of the most sensitive human malignancies was found to be childhood acute lymphoblastic leukemia, and asparaginase is now routinely used in induction and consolidation protocols for patients with this disease (23). In mouse model systems, it was found that asparaginase-sensitive tumor cells could be made asparaginase-resistant by repeated subculturing of these cells in sublethal concentrations of the drug. The resistant cells were found to have regained AS activity and in some cases had elevated levels of enzyme activity up to 60-fold higher than that in normal cells (13,24). The mechanism responsible for the lack of activity of AS in the sensitive leukemic cells and for the overproduction in the asparaginase-resistant variants is of clinical importance for the treatment of acute lymphoblastic leukemia patients both initially and during relapse.To investigate the molecular basis for the sensitivity to asparaginase in human acute...

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

“…Ec-AsnB The alfalfa AS (Ms-AS) sequence was aligned with AS sequences from Pisum sativum (Ps-ASI; GenBank accession number X52179; Tsai and Coruzzi, 1990), Arabidopsis tbaliana (At-ASN1 ; GenBank accession number L29083; Lam et al, 1994), Zea mays (Zm-AS; GenBank accession number X82849; Chevalier et al, 1996), humans (Hum-AS; GenBank accession number M27396; Andrulis et al, 1987), yeast (Sc-ASNI; GenBank accession number U40829), and E. coli (Ec-AsnB; GenBank accession number J05554; Scofield et al, 1990). ldentical residues are indicated by dots.…”

Overexpression and Purification of Asparagine Synthetase fromEscherichia coli

“…9 ,11) Both types of asparagine synthetase genes have been cloned and sequenced from E. COli 12 ,13) and humans. 4 ) The asnA gene from E. coli coded for a polypeptide of 330 amino acid residues with a molecular weight of 36,700. A high degree of amino acid sequence homology was shown between the glutamine-dependent enzymes (asnB enzyme from E. coli and the human enzyme), however, no significant homology was found between the asnA and the asnB sequences although derived from the same origin, E. coli.…”

Asparagine synthetase gene expression increases as sucrose declines in broccoli after harvest

“…The predicted amino acid sequence of this broccoli AS cDNA has 84% identity to the AS polypeptide from asparagus (Davies & King 1993), and contains a glutaminebinding motif at the N-terminus in common with the human and other plant AS sequences (Andrulis et al 1987;van Heeke & Schuster 1989;Davies & King 1993). In broccoli Northern analyses reported here, we detect transcripts of c. 2 000 bp, similar to the size detected in asparagus AS Northerns.…”