Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells

1983 ◽  
Vol 3 (3) ◽  
pp. 391-398
Author(s):  
I L Andrulis ◽  
C Duff ◽  
S Evans-Blackler ◽  
R Worton ◽  
L Siminovitch
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Asparagine Synthetase ◽  
Chromosome 1 ◽  
Ovary Cell ◽  
Synthetase Activity

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.

scholarly journals Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells.

1983 ◽  
Vol 3 (3) ◽  
pp. 391-398 ◽  
Author(s):  
I L Andrulis ◽  
C Duff ◽  
S Evans-Blackler ◽  
R Worton ◽  
L Siminovitch
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Asparagine Synthetase ◽  
Chromosome 1 ◽  
Ovary Cell ◽  
Synthetase Activity

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

1989 ◽  
Vol 9 (7) ◽  
pp. 2922-2927
Author(s):  
I L Andrulis ◽  
M T Barrett
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary Cells ◽  
Methylation Status ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Methylation Pattern ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Dna Hypomethylation ◽  
Ovary Cells

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.

scholarly journals DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

1989 ◽  
Vol 9 (7) ◽  
pp. 2922-2927 ◽  
Author(s):  
I L Andrulis ◽  
M T Barrett
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary Cells ◽  
Methylation Status ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Methylation Pattern ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Dna Hypomethylation ◽  
Ovary Cells

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.

Amplification of the gene for histidyl-tRNA synthetase in histidinol-resistant Chinese hamster ovary cells

1985 ◽  
Vol 5 (9) ◽  
pp. 2381-2388
Author(s):  
F W Tsui ◽  
I L Andrulis ◽  
H Murialdo ◽  
L Siminovitch
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Wild Type ◽  
Ovary Cells ◽  
Resistant Cells

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

Paraquat-resistant cell lines derived from chinese hamster ovary cells

1990 ◽  
Vol 14 (3) ◽  
pp. 235-246 ◽  
Author(s):  
T LEE ◽  
F LIN ◽  
I HO ◽  
T LIU ◽  
T WANG ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Ovary Cells

scholarly journals Isolation of Chinese hamster ovary cell lines temperature conditional for the cell-surface expression of integral membrane glycoproteins.

1989 ◽  
Vol 108 (2) ◽  
pp. 339-353 ◽  
Author(s):  
J Hearing ◽  
E Hunter ◽  
L Rodgers ◽  
M J Gething ◽  
J Sambrook
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Ovary Cell ◽  
Cell Surface Expression ◽  
Membrane Glycoproteins

A procedure is described to select mutants of Chinese hamster ovary cells that are conditionally defective for the cell-surface expression of integral membrane glycoproteins, including the hemagglutinin (HA) of influenza virus. Using a combination of cell sorting and biochemical screening, seven cell lines were obtained that express more cell-surface HA at 32 degrees C than at 39 degrees C. The production of infectious vesicular stomatitis virus, whose growth requires insertion of an integral membrane protein into the plasma membrane, was also temperature conditional in the majority of these mutant cell lines. Five of the lines synthesized apparently normally core-glycosylated HA at the elevated temperature but the protein was neither displayed on the cell surface nor accumulated intracellularly. In these cell lines, little or no terminally glycosylated HA molecules were observed after synthesis at 39 degrees C. By contrast, the core glycosylation of HA and several other integral membrane proteins was abnormal in the remaining two cell lines at both permissive and restrictive temperatures, due to a lesion in a cellular gene(s) that affects the formation of and/or the addition of mannose-rich oligosaccharide chains to newly synthesized polypeptides. Although HA was transported to the plasma membrane at both 32 and 39 degrees C, it did not accumulate on the cell surface at the higher temperature, apparently because of an increased rate of degradation.

scholarly journals Amplification of the gene for histidyl-tRNA synthetase in histidinol-resistant Chinese hamster ovary cells.

1985 ◽  
Vol 5 (9) ◽  
pp. 2381-2388 ◽  
Author(s):  
F W Tsui ◽  
I L Andrulis ◽  
H Murialdo ◽  
L Siminovitch
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Wild Type ◽  
Ovary Cells ◽  
Resistant Cells

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

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