Concanavalin A as a selective agent in tissue culture for temperature-sensitive hamster cell lines

1975 ◽  
Vol 21 (10) ◽  
pp. 1650-1654 ◽  
Author(s):  
Jim A. Wright
Keyword(s):  
Tissue Culture ◽  
Cell Lines ◽  
Concanavalin A ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Resistant Cell ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Selective Agent ◽  
Growth Properties

A novel method for isolating Chinese hamster cell cultures with temperature-sensitive growth properties is described. Concanavalin A can be used as a selective agent in tissue culture to isolate lectin-resistant cell lines which exhibit colony-forming abilities at the nonpermissive temperature relative to the permissive temperature as low as 10−4 to 10−6. A general correlation exists between resistance to the lectin and temperature sensitivity.

Growth Properties of Neural Cell Lines Immortalized with the Tsa58 Allele of Sv40 Large T Antigen

1997 ◽  
Vol 6 (3) ◽  
pp. 231-238 ◽  
Author(s):  
M.E. Truckenmiller ◽  
Ora Dillon-Carter ◽  
Carlo Tornatore ◽  
Henrietta Kulaga ◽  
Hidetoshi Takashima ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Cell Lines ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Large T Antigen ◽  
Wild Type ◽  
Sv40 Large T Antigen ◽  
Growth Properties

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33° C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5°C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5°C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5°C for all three cell lines. After 3 wk at 39.5°C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure

1988 ◽  
Vol 8 (12) ◽  
pp. 5268-5279
Author(s):  
J E Looney ◽  
C Ma ◽  
T H Leu ◽  
W F Flintoff ◽  
W B Troutman ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Resistant Cell ◽  
Pulse Field ◽  
Ovary Cell ◽  
Type I

We have previously cloned and characterized two different dihydrofolate reductase amplicon types from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). The largest of these (the type I amplicon) is 273 kilobases (kb) in length. In the present study, we utilized clones from the type I amplicon as probes to analyze the size and variability of the amplified DNA sequences in five other independently isolated methotrexate-resistant Chinese hamster cell lines. Our data indicated that the predominant amplicon types in all but one of these cell lines are larger than the 273-kb type I sequence. In-gel renaturation experiments as well as hybridization analysis of large SfiI fragments separated by pulse-field gradient gel electrophoresis showed that two highly resistant cell lines (A3 and MK42) have amplified very homogeneous core sequences that are estimated to be at least 583 and 653 kb in length, respectively. Thus, the sizes of the major amplicon types can be different in different drug-resistant Chinese hamster cell lines. However, there appears to be less heterogeneity in size and sequence arrangement within a given methotrexate-resistant Chinese hamster cell line than has been reported for several other examples of DNA sequence amplification in mammalian systems.

scholarly journals Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.

1983 ◽  
Vol 3 (5) ◽  
pp. 892-902 ◽  
Author(s):  
R E Cirullo ◽  
S Dana ◽  
J J Wasmuth
Keyword(s):  
Human Genome ◽  
Cell Lines ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Efficient Procedure ◽  
Human Dna ◽  
Human Genes ◽  
Genes Encoding

We have developed a simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, we have constructed Chinese hamster cell lines that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure. Analysis of these cell lines with Southern blots confirmed the presence of a small number of restriction endonuclease fragments containing human DNA specifically. These cell lines represent a convenient and simple means to clone the human genomic sequences of interest.

scholarly journals The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure.

1988 ◽  
Vol 8 (12) ◽  
pp. 5268-5279 ◽  
Author(s):  
J E Looney ◽  
C Ma ◽  
T H Leu ◽  
W F Flintoff ◽  
W B Troutman ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Resistant Cell ◽  
Pulse Field ◽  
Ovary Cell ◽  
Type I

We have previously cloned and characterized two different dihydrofolate reductase amplicon types from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). The largest of these (the type I amplicon) is 273 kilobases (kb) in length. In the present study, we utilized clones from the type I amplicon as probes to analyze the size and variability of the amplified DNA sequences in five other independently isolated methotrexate-resistant Chinese hamster cell lines. Our data indicated that the predominant amplicon types in all but one of these cell lines are larger than the 273-kb type I sequence. In-gel renaturation experiments as well as hybridization analysis of large SfiI fragments separated by pulse-field gradient gel electrophoresis showed that two highly resistant cell lines (A3 and MK42) have amplified very homogeneous core sequences that are estimated to be at least 583 and 653 kb in length, respectively. Thus, the sizes of the major amplicon types can be different in different drug-resistant Chinese hamster cell lines. However, there appears to be less heterogeneity in size and sequence arrangement within a given methotrexate-resistant Chinese hamster cell line than has been reported for several other examples of DNA sequence amplification in mammalian systems.

Bmps(Bone Morphogenetic Proteins) Inhibit Growth in Multiple Myeloma Cells by p53 Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3354-3354
Author(s):  
Anders Sundan ◽  
Torstein B. Ro ◽  
Janne Bonhorst ◽  
Anders Waage ◽  
Magne Borset
Keyword(s):  
Multiple Myeloma ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Target Genes ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
P53 Mutation ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Myeloma Cells

Abstract Only a few naturally occurring cytokines are able to inhibit myeloma cell growth, and among them BMP-4 is a potent inhibitor of growth as well as an inductor of apoptosis in myeloma cells. To study the mechanism behind BMP-4-induced growth inhibition, we performed gene expression profiling by microarrays in two human myeloma cell lines after BMP-4 stimulation for 4 hours. We found that BMP-4 upregulated several known p53 target genes like p21/Cip1, Bax, cyclin G, Gadd45 and dual specificity phosphatases, suggesting a role for p53 in BMP-mediated growth inhibition of myeloma cells. p53 is activated by several post-translational modifications, including phosphorylation at several serine/threonine residues. We found that BMP-4 treatment lead to rapid phosphorylation of p53 at Serine 15. Furthermore, we were able to co-precipitate phosphorylated Smad-1/5/8 and p53 from nuclear extracts of cells treated with BMP by applying a p53 binding element from the Gadd45 promoter. Because some myeloma cell lines are resistant for BMP-4-induced growth inhibition, we examined whether this BMP-4 resistance was associated with p53 mutations in the BMP-4-resistant cell lines. We sequenced the p53 cDNA from eight myeloma cell lines, and resistant cells all had mutated or deleted p53. Introduction of normal p53 by adenoviral constructs in the p53 mutated cell lines restored BMP-4 responsiveness to some degree. Furthermore, in a myeloma cell line with a temperature-sensitive p53 mutation, the cells were sensitive towards BMP-induced growth inhibition at the permissive temperature but resistant when p53 was inactive. In conclusion, we show that normal p53 activity is involved in BMP-4-induced growth inhibition of multiple myeloma cells.

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

1983 ◽  
Vol 3 (5) ◽  
pp. 892-902
Author(s):  
R E Cirullo ◽  
S Dana ◽  
J J Wasmuth
Keyword(s):  
Human Genome ◽  
Cell Lines ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Efficient Procedure ◽  
Human Dna ◽  
Human Genes ◽  
Genes Encoding

We have developed a simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, we have constructed Chinese hamster cell lines that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure. Analysis of these cell lines with Southern blots confirmed the presence of a small number of restriction endonuclease fragments containing human DNA specifically. These cell lines represent a convenient and simple means to clone the human genomic sequences of interest.

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.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity

1983 ◽  
Vol 3 (6) ◽  
pp. 1053-1061
Author(s):  
W H Lewis ◽  
P R Srinivasan
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Plating Efficiency ◽  
Wild Type Cell ◽  
Wild Type ◽  
Metaphase Chromosomes

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.

Chinese Hamster Cell Variants Resistant to the A Chain of Ricin Carry Altered Ribosome Function

1982 ◽  
Vol 2 (6) ◽  
pp. 599-606
Author(s):  
Mayumi Ono ◽  
Michihiko Kuwano ◽  
Kei-Ichi Watanabe ◽  
Gunki Funatsu
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Cho Cells ◽  
Ricinus Communis ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Cytotoxic Action ◽  
A Chain ◽  
Ricin A

Ricin, a toxic lectin from Ricinus communis , is composed of two different polypeptide chains, A and B, and the ricin A chain (RA) blocks protein synthesis. We studied cell lines resistant to cytotoxic action of RA. One low-RA-resistant cell line, AR10, isolated from Chinese hamster ovary (CHO) cells, was resistant to a low dose of RA (1 μg/ml) and showed a 10-fold-higher resistance to RA and ricin than that of CHO. We further mutagenized AR10 to isolate high-RA-resistant cell lines AR100-6, AR100-9, and AR100-13, which were resistant to higher doses of RA and ricin (100- to 1,000-fold) than CHO was. The binding of [ 125 I]ricin to AR10, AR100-6, AR100-9, and AR100-13 cells was decreased to about 30% of that of CHO. The internalization of [ 125 I]ricin in AR10 cells and in the high-RA-resistant clones was the same. Polyuridylate-dependent polyphenylalanine synthesis, using S-30 extracts from either AR100-9 or AR100-13, was about 100-fold more resistant to the inhibitory action of RA than when CHO, AR10, and AR100-6 cells extracts were used. The protein synthesis with ribosomes (80S) from AR100-9 or AR100-13 was 10- to 100-fold more resistant to RA than it was with parental ribosomes when combined with the S-100 fraction of CHO cells. The polyphenylalanine synthesis assay using the ribosomes constituted from the 60S subunit of AR100-9 and the 40S subunit of CHO indicated that the resistant phenotype of AR100-9 cells is due to an alteration of the 60S ribosomal subunit.

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