Characterization of single step albizziin-resistant Chinese hamster ovary cell lines with elevated levels of asparagine synthetase activity.

DNA Methylation Patterns Associated with Asparagine Synthetase Expression in Asparagine-Overproducing and -Auxotrophic Cells

“…These included changes in K,,, and Kj, differences in cross-resistance to 3-aspartyl hydroxamate, and alterations in AS activity in response to the concentration of asparagine in the medium. However, all of the single-step AlbR mutants produced levels of AS enzymatic activity 8to 17-fold greater than those of the parental lines (3). The molecular basis for overproduction of AS enzymatic activity in the mutants was not determined, although we did show that one of the single-step AlbR mutants contained amplified copies of the gene for AS (25).…”

“…We have previously described biochemical studies on AS regulation (4) and genetic characterization of AlbR and I-AHAR cells that overproduce AS activity (2,3,6). We have begun a molecular analysis of the mutant lines and found that AlbR cells have amplified copies of the gene for AS, whereas P-AHAR cell lines with high levels of resistance have no increase in AS copy number.…”

“…We have also investigated the role of DNA methylation in the expression of AS activity in drug-resistant mutant cell lines that overproduce the enzyme (2,3,6). Single-step mutant lines resistant to the amino acid analog P-aspartyl hydroxamate (P-AHAR) exhibit elevations in AS activity up to 8-fold over that of the parental cells (6,11), and multistep 1-AHAR lines express as high as 20-fold increases in AS activity (6).…”

“…MATERIALS AND METHODS Cell lines. Isolation of the AlbR CHO mutant lines has been previously described (2,3). Wild-type (Toronto strain) CHO cells and single-step AlbR mutants (AlbR 1, 2, 6, 10, and 27) were grown in a complete (ot minimal essential medium [28] containing nucleosides and 7% heat-inactivated [56°C for 30 min] fetal calf serum).…”

Isolation of Human cDNAs for Asparagine Synthetase and Expression in Jensen Rat Sarcoma Cells

“…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).…”