Chromosomal Alterations Associated with Overproduction of Asparagine Synthetase in Albizziin-Resistant Chinese Hamster Ovary Cells

Abstract

The amino acid analog albizziin was used to isolate Chinese hamster ovary cell lines which overproduce asparagine synthetase. Mutants selected in a single step after ethyl methane sulfonate mutagenesis were approximately 10-fold more resistant to the drug than the parental lines and expressed 8- to 17-fold elevations in enzyme activity. The karyotypes of these lines show alterations such as breaks and translocations affecting the long arm of chromosome 1. Cell lines isolated in several steps by growth in progressively increasing concentrations of albizziin were more resistant to the drug and exhibited up to 300-fold enhancement of asparagine synthetase activity. The multistep albizziin-resistant cell lines usually had expanded chromosomal regions which stained somewhat homogeneously, often on the long arm of chromosome 1. These results suggest that resistance to albizziin in the multistep lines may be due to gene amplification.

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

“…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).…”

“…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). On the other hand multistep albizziin-resistant (AlbR) lines that overproduce the enzyme by 300-fold have been isolated (2). We took advantage of a highly resistant multistep line to clone an AS cDNA and show that the gene was amplified in this line (25).…”

“…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).…”

Unstable and Stable CAD Gene Amplification: Importance of Flanking Sequences and Nuclear Environment in Gene Amplification

“…One aspect of the amplification process which varies from cell line to cell line is whether the amplified sequences are localized to a chromosome or are found extrachromosomally. For example, although exceptions exist (2,12,20), amplification in mouse cells tends to be extrachromosomal, whereas it is usually within chromosomes in hamster cells (for reviews, see references 6, 8, 19, and 39). While it is generally believed that some feature of the different cell lines in which gene amplification has been observed determines the cytogenetic outcome of the amplification process, this conclusion is mainly based on a comparison of the results obtained for the amplification of different genes in different cell lines.…”