Chinese hamster ovary cell mutants specifically affected in the phosphorylation of C-purine nucleosides

1985 ◽  
Vol 63 (9) ◽  
pp. 1044-1048 ◽  
Author(s):  
Kamal D. Mehta ◽  
Radhey S. Gupta
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Nucleoside Analogs ◽  
Adenosine Kinase ◽  
Ovary Cell ◽  
Cross Resistance ◽  
Ovary Cells ◽  
C And N ◽  
High Degree

In Chinese hamster ovary cells, two different types of mutants resistant to purine nucleoside analogs have been isolated. One type of mutants selected in presence of C-nucleosides formycin A and formycin B (FomR mutants) exhibited a high degree of cross resistance to different C-nucleosides but showed very slight to no cross resistance to various N-nucleosides. In contrast, mutants selected in presence of toyocamycin (Toyr mutants) exhibited a high degree of cross resistance to all C- and N-adenosine analogs. Studies on the cellular uptake and phosphorylation of [3H]adenosine, [3H]tubercidin, and [3H]formycin A show that, unlike the Toyr mutants which show reduced phosphorylation of all three compounds, the FomR mutants show reduced cellular phosphorylation of only [3H]formycin A. It is suggested that the genetic lesion in the FomR mutants affects adenosine kinase in a novel manner that specifically affects the phosphorylation of C-purine nucleosides.

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase

1983 ◽  
Vol 3 (8) ◽  
pp. 1468-1477
Author(s):  
K D Mehta ◽  
R S Gupta
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Form ◽  
Single Step ◽  
Adenosine Kinase ◽  
Cross Resistance ◽  
Ovary Cells ◽  
Cell Extracts

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

scholarly journals Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

1983 ◽  
Vol 3 (8) ◽  
pp. 1468-1477 ◽  
Author(s):  
K D Mehta ◽  
R S Gupta
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Form ◽  
Single Step ◽  
Adenosine Kinase ◽  
Cross Resistance ◽  
Ovary Cells ◽  
Cell Extracts

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer

1989 ◽  
Vol 9 (4) ◽  
pp. 1754-1758
Author(s):  
T M Underhill ◽  
W F Flintoff
Keyword(s):  
Gene Transfer ◽  
Dna Sequences ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Ovary Cells ◽  
Ovary Cell Line ◽  
Human Specific

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.

Chinese hamster ovary cell mutants defective in the internalization of ricin

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

scholarly journals Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line.

1981 ◽  
Vol 1 (12) ◽  
pp. 1069-1076 ◽  
Author(s):  
R J Kaufman ◽  
R T Schimke
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Ovary Cells ◽  
Methotrexate Resistance ◽  
Gene Copies ◽  
Methotrexate Concentration ◽  
Ovary Cell Line

During stepwise increases in the methotrexate concentration in culture medium, we selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). We studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

scholarly journals Selection of specific wheat germ agglutinin-resistant (WgaR) phenotypes from Chinese hamster ovary cell populations containing numerous lecR genotypes.

1981 ◽  
Vol 1 (8) ◽  
pp. 687-696 ◽  
Author(s):  
P Stanley
Keyword(s):  
Chinese Hamster Ovary ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Ovary Cell ◽  
Mutant Type ◽  
Similar Frequency ◽  
Ovary Cells

Three distinct Chinese hamster ovary mutants selected for resistance to wheat germ agglutinin were previously described by this laboratory. In this paper, evidence is provided that each phenotype occurs at a similar frequency in an unmutagenized population of Chinese hamster ovary cells. Two novel wheat germ agglutinin resistance phenotypes (WgaR), which also appear to occur at similar frequencies were uncovered in the course of these studies. One mutant type belongs to a new, recessive complementation group (VIII), and the second belongs to a previously defined complementation group (VI). Mutants from each of the four WgaR complementation groups (I, II, III, and VIII) exhibited characteristic and unique patterns of resistance to the toxicity of a variety of plant lectins. These properties were used in developing independent selection protocols which were highly specific for the isolation of each of the mutant types.

Melanogenesis in murine B16 cells exposed to Aeromonas hydrophila cytotoxic enterotoxin

1986 ◽  
Vol 32 (10) ◽  
pp. 814-819 ◽  
Author(s):  
V. K. Bunning ◽  
R. G. Crawford ◽  
G. N. Stelma Jr. ◽  
L. O. Kaylor ◽  
C. H. Johnson
Keyword(s):  
Cholera Toxin ◽  
Chinese Hamster Ovary ◽  
Aeromonas Hydrophila ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Transferase Activity ◽  
End Points ◽  
Ovary Cells ◽  
Culture Filtrates

Specific markers (growth, melanogenesis) of B16 murine melanoma cells in culture were used as indicators of toxin production by Aeromonas hydrophila. Cytotonic enterotoxinlike activity (inhibited growth, raised tyrosinase activity, and melanin accumulation) occurred at cytotoxic end points of purified β-hemolysin and several culture filtrates. Antihemolysin rabbit serum inhibited this activity. A hemolysin-neutralized culture filtrate concentrate (10×) failed to elevate tyrosinase relative to untreated and cholera toxin treated controls. Similar dilution profiles using Chinese hamster ovary cells showed limited cell extension only at cytotoxic end points with antihemolysin inhibiting this activity. Cytotoxicity of Chinese hamster ovary cells and B16 cells was proportional to hemolytic activity, with B16 cells showing about 100-fold greater sensitivity on a per cell basis. Cell culture cytotoxicity attributed to β-hemolysin correlated with reactivity in rabbit ileal loop assays. The ADP-ribosyl transferase activity of concentrated (10×) A. hydrophila culture filtrates and fractions thereof was negative. Apparently sublethal doses of A. hydrophila β-hemolysin can nonspecifically stimulate cyclic adenosine monophosphate mediated events in melanoma and Chinese hamster ovary cell assays, producing lower activities than cholera toxin with shorter lag times.

scholarly journals Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

1984 ◽  
Vol 4 (2) ◽  
pp. 296-301 ◽  
Author(s):  
B Storrie ◽  
M Sachdeva ◽  
V S Viers
Keyword(s):  
Horseradish Peroxidase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Ovary Cell ◽  
Cell Fractionation ◽  
Marker Enzyme ◽  
Ovary Cells

We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

scholarly journals Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

1982 ◽  
Vol 2 (3) ◽  
pp. 250-257 ◽  
Author(s):  
J A Tischfield ◽  
J J Trill ◽  
Y I Lee ◽  
K Coy ◽  
M W Taylor
Keyword(s):  
Chinese Hamster Ovary ◽  
Hot Spot ◽  
Chinese Hamster Ovary Cells ◽  
Human Fibroblasts ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Adenine Phosphoribosyltransferase ◽  
Ovary Cells ◽  
L Cells ◽  
A Site

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.

Sign in / Sign up

Export Citation Format

Share Document