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

Download Full-text

Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.1832-1838.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 1832-1838

Author(s):

K J Kontis ◽

S M Arfin

Keyword(s):

Cdna Clone ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Mrna Translation ◽

Resistant Cell ◽

Trna Synthetase ◽

Expression Library ◽

Hybridization Probe ◽

Ovary Cells

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA lambda gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.

Download Full-text

Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells

Canadian Journal of Biochemistry ◽

10.1139/o77-062 ◽

1977 ◽

Vol 55 (4) ◽

pp. 445-452 ◽

Cited By ~ 27

Author(s):

Radhey S. Gupta ◽

Wayne F. Flintoff ◽

Louis Siminovitch

Keyword(s):

Dihydrofolate Reductase ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Class I ◽

Class Iii ◽

Wild Type ◽

Ovary Cells ◽

I Cells ◽

Resistant Cells

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levels of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W. F., Davidson, S. V., and Siminovitch, L. (1976) Somatic Cell Genet. 2, 245–261). By selecting for increased resistance from these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In this report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and a class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.

Download Full-text

Selection of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosaminyltransferase activity.

The Journal of Cell Biology ◽

10.1083/jcb.94.3.586 ◽

1982 ◽

Vol 94 (3) ◽

pp. 586-591 ◽

Cited By ~ 27

Author(s):

B A Criscuolo ◽

S S Krag

Keyword(s):

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Fold Increase ◽

Transferase Activity ◽

Wild Type ◽

Vitro Assay ◽

Glycoprotein Biosynthesis ◽

Resistant Cells

Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) have been isolated by a stepwise selection procedure with progressive increments of TM added to the medium. TM inhibits asparagine-linked glycoprotein biosynthesis by blocking the transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the lipid carrier. The TM-resistant cells exhibited a 200-fold increase in their LD50 for TM and were morphologically distinct from the parental cells. The rate of asparagine-linked glycoprotein biosynthesis was the same for wild-type and TM-resistant cells. Membrane preparations from TM-resistant cells cultured for 16 d in the absence of TM had a 15-fold increase in the specific activity of the UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase as compared to membranes of wild-type cells. The products of the in vitro assay were N-acetylglucosaminylpyrophosphoryl-lipid and N,N'-diacetylchitobiosylpyrophosphoryl-lipid for membranes from both TM-resistant and wild-type cells. The transferase activity present in membrane preparations from wild-type of TM-resistant cells was inhibited by comparable levels of TM. The data presented are consistent with overproduction of enzyme as the mechanism of resistance in these variant CHO cells.

Download Full-text

Characterization of cell lines showing growth control isolated from both the wild type and a leucyl-tRNA synthetase mutant of chinese hamster ovary cells

Journal of Cellular Physiology ◽

10.1002/jcp.1040980315 ◽

1979 ◽

Vol 98 (3) ◽

pp. 571-585 ◽

Cited By ~ 64

Author(s):

J. W. Pollard ◽

C. P. Stanners

Keyword(s):

Cell Lines ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Growth Control ◽

Trna Synthetase ◽

Wild Type ◽

Ovary Cells

Download Full-text

Cytoplasmic inheritance of oligomycin resistance in Chinese hamster ovary cells.

The Journal of Cell Biology ◽

10.1083/jcb.86.3.723 ◽

1980 ◽

Vol 86 (3) ◽

pp. 723-729 ◽

Cited By ~ 16

Author(s):

G A Breen ◽

I E Scheffler

Keyword(s):

Chinese Hamster Ovary ◽

Doubling Time ◽

Chinese Hamster Ovary Cells ◽

Mitochondrial Protein ◽

Chinese Hamster ◽

Fold Increase ◽

Cross Resistance ◽

Mitochondrial Atpase ◽

Wild Type ◽

Ovary Cells

Oligomycin-resistant clones were isolated from Chinese hamster ovary cells by treatment of cells with ethidium bromide, followed by mutagenesis with ethylmethane sulfonate and selection in oligomycin. One clone (Olir 8.1) was chosen for further study. Olir 8.1 cells grow with doubling time similar to that of wild-type cells, whether grown in the presence or absence of drug (doubling time of 13-14 h). In plating efficiency experiments, Olir 8.1 cells are approximately 100-fold more resistant to oligomycin than are wild-type cells. There is approximately a 32-fold increase in the resistance to inhibition by oligomycin of the mitochondrial ATPase from Olir 8.1 cells. The electron transport chain is functional in Olir 8.1 cells. Oligomycin resistance is stable in the absence of selective pressure. There is little or no cross-resistance of Olir 8.1 cells to venturicidin and dicyclohexylcarbodiimide, other inhibitors of the mitochondrial ATPase, or to chloramphenicol, an inhibitor of mitochondrial protein synthesis. Oligomycin resistance is dominant in hybrids between Olir 8.1 cells and wild-type cells. Fusions of enucleated Olir 8.1 cells with sensitive cells and characterization of the resulting "cybrid" clones indicates that oligomycin resistance in Olir 8.1 cells is cytoplasmically inherited.

Download Full-text

Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines.

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.1832 ◽

1989 ◽

Vol 9 (5) ◽

pp. 1832-1838 ◽

Cited By ~ 7

Author(s):

K J Kontis ◽

S M Arfin

Keyword(s):

Cdna Clone ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Mrna Translation ◽

Resistant Cell ◽

Trna Synthetase ◽

Expression Library ◽

Hybridization Probe ◽

Ovary Cells

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA lambda gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.

Download Full-text