Selection of temperature-resistant mutants of Chinese hamster cells for growth on D-galactose

1982 ◽  
Vol 28 (3) ◽  
pp. 356-359
Author(s):  
Jean-Paul Thirion ◽  
E. J. Aw
Keyword(s):  
Gene Regulation ◽  
Mammalian Cells ◽  
Selection Pressure ◽  
Chinese Hamster ◽  
Wild Type ◽  
Chinese Hamster Cells ◽  
Growth Properties ◽  
Regulation Of Carbohydrate Metabolism ◽  
Resistant Mutants ◽  
Selection Of

Somatic cell variants of Chinese hamster V79 were selected for rapid growth on D-galactose at high temperatures. Their phenotypes are stable after many generations in the absence of selection pressure. They clone in D-galactose at temperatures at which the wild type cannot, while in D-glucose, the variants and the wild type appear to have the same growth properties. The use of such variants should be very important for the study of gene regulation of carbohydrate metabolism in mammalian cells.

scholarly journals 8-AZAGUANINE RESISTANCE IN MAMMALIAN CELLS I. HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE

Genetics ◽  
1972 ◽  
Vol 72 (2) ◽  
pp. 239-252 ◽  
Author(s):  
F D Gillin ◽  
D J Roufa ◽  
A L Beaudet ◽  
C T Caskey
Keyword(s):  
Nucleic Acid ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Acid Synthesis ◽  
Ethyl Methanesulfonate ◽  
Nucleic Acid Synthesis ◽  
Wild Type ◽  
Chinese Hamster Cells ◽  
Hypoxanthine Guanine Phosphoribosyltransferase

ABSTRACT Chinese hamster cells were treated with ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine, and mutants resistant to 8-azaguanine were selected and characterized. Hypoxanthine-guanine phosphoribosyltransferase activity of sixteen mutants is extremely negative, making them suitable for reversion to HGPRTase+. Ten of the extremely negative mutants revert at a frequency higher than 10-7 suggesting their point mutational character. The remaining mutants have demonstrable HGPRTase activity and are not useful for reversion analysis. Five of these mutants have < 2% HGPRTase and are presumably also HGPRTase point mutants. The remaining 14 mutants utilize exogenous hypoxanthine for nucleic acid synthesis poorly, and possess 20-150% of wild-type HGPRTase activity in in vitro. Their mechanism of 8-azaguanine resistance is not yet defined.

scholarly journals DERIVATION OF TK- CLONES FROM REVERTANT TK+ MAMMALIAN CELLS

Genetics ◽  
1973 ◽  
Vol 75 (3) ◽  
pp. 515-530
Author(s):  
D J Roufa ◽  
B N Sadow ◽  
C T Caskey
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Single Step ◽  
Wild Type ◽  
Cell Clones ◽  
Selection Experiments ◽  
Step Selection ◽  
Selection Of

ABSTRACT In order to obtain a large collection of Chinese hamster cell clones defective in thymidine kinase (TK-), BrdUr selection experiments have been performed on wild-type and revertant TK+ cell lines. No clones (< 10-9) were obtained from the wild-type TK+ cell line by single-step selection. In contrast, revertant TK+ clones readily gave rise to stable TK- derivatives (1 - 2 × 10-4). Both wild-type and revertant TK+ clones spontaneously yielded 8-AGr colonies with the same frequency (1 - 5 × 10-6), suggesting that the differences between wild-type and revertant cell lines specifically affected selection of the TK- phenotype. The increased frequency of TK- clones reflects perhaps the number (ploidy) or character of the autosomal TK loci in TK+ revertants, or perhaps the mechanisms which regulate expression of the TK genes. Several mutagens, EMS, MNNG and UV, stimulated the TK+ revertants' frequency of TK- subclones only slightly (< 3-fold). Biochemical and genetic data indicated that the TK- clones derived from one revertant are phenotypically different. The phenotypes displayed by these cell lines are stable and do not depend upon the continued presence of the selective agent.

Correction of Genetic Defects in Mammalian Cells by the Input of Small Amounts of Foreign Genetic Material

1973 ◽  
Vol 13 (3) ◽  
pp. 841-861
Author(s):  
YVONNE L. BOYD ◽  
H. HARRIS
Keyword(s):  
Thymidine Kinase ◽  
Mammalian Cells ◽  
Hybrid Cell ◽  
Genetic Material ◽  
Chinese Hamster ◽  
Heat Sensitivity ◽  
Chinese Hamster Cells ◽  
Inosinic Acid ◽  
Mouse Cells

Chinese hamster cells lacking inosinic acid pyrophosphorylase and mouse cells lacking thymidine kinase were fused with chick erythrocytes. The resultant heterokaryons were cultivated in a selective medium in which possession of these enzymes was essential for cell survival and growth. Clones of cells able to grow in this medium were isolated and studied. A detailed karyological analysis of these clones failed to reveal any chick chromosomes; nor could any chick-specific antigens be detected on the surface of the cells. Nonetheless, clones arising from the fusion of chick erythrocytes with Chinese hamster cells were shown to possess an inosinic acid pyrophosphorylase which had the electrophoretic characteristics of chick inosinic acid pyrophosphorylase. However, the clones arising from the fusion of the chick erythrocytes with the mouse cells had a thymidine kinase with the electrophoretic mobility and heat sensitivity of murine, not chick, thymidine kinase. Both types of hybrid cell have now been cultivated in vitro for 18 months without the loss of thymidine kinase or inosinic acid pyrophosphorylase activity.

scholarly journals STUDIES OF DNA-INDUCED HERITABLE ALTERATION OF MAMMALIAN CELLS

1970 ◽  
Vol 132 (6) ◽  
pp. 1071-1089 ◽  
Author(s):  
Ellen Borenfreund ◽  
Yuji Honda ◽  
Mildred Steinglass ◽  
Aaron Bendich
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster ◽  
Ascites Tumor ◽  
Intercellular Interaction ◽  
Oncogenic Potential ◽  
Chinese Hamster Cells ◽  
Direct Microscopic Examination ◽  
Ehrlich Ascites ◽  
Specific Antigens

An intercellular interaction between mouse Ehrlich ascites tumor and non-malignant Chinese hamster cells occurred when these were co-cultured. That the intercellular processes which formed had emanated from the EA cells was revealed by immunofluoroscopy using anti-EA antiserum, and by direct microscopic examination. A passage of DNA from the EA to the CH cells was also observed. On long-term co-culture, new cell forms arose which were isolated, cloned, and propagated. They showed a CH karyotype and had acquired oncogenic potential and the ability to synthesize murine-specific antigens. These same heritable properties were also acquired by CH cells following their exposure to DNA isolated from EA cells.

scholarly journals Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone

2002 ◽  
Vol 46 (11) ◽  
pp. 3370-3380 ◽  
Author(s):  
Dilek Ince ◽  
Xiamei Zhang ◽  
L. Christine Silver ◽  
David C. Hooper
Keyword(s):  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Fold Increase ◽  
Single Step ◽  
The Novel ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dual Targeting ◽  
Resistant Mutants ◽  
Selection Of

ABSTRACT We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 μg/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.

scholarly journals Selection of Retroviral Reverse Transcription Primer Is Coordinated with tRNA Biogenesis

2003 ◽  
Vol 77 (16) ◽  
pp. 8695-8701 ◽  
Author(s):  
Nathan J. Kelly ◽  
Matthew T. Palmer ◽  
Casey D. Morrow
Keyword(s):  
Reverse Transcription ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Leukemia Virus ◽  
Wild Type ◽  
Primer Selection ◽  
Yeast Trna ◽  
Trna Primer ◽  
Hiv 1 ◽  
Selection Of

ABSTRACT Initiation of retrovirus reverse transcription requires the selection of a tRNA primer from the intracellular milieu. To investigate the features of primer selection, a human immunodeficiency virus type 1 (HIV-1) and a murine leukemia virus (MuLV) were created that require yeast tRNAPhe to be supplied in trans for infectivity. Wild-type yeast tRNAPhe expressed in mammalian cells was transported to the cytoplasm and aminoacylated. In contrast, tRNAPhe without the D loop (tRNAPheD−) was retained within the nucleus and did not complement infectivity of either HIV-1 or MuLV; however, infectivity was restored when tRNAPheD− was directly transfected into the cytoplasm of cells. A tRNAPhe mutant (tRNAPheUUA) that did not have the capacity to be aminoacylated was transported to the cytoplasm and did complement infectivity of both HIV-1 and MuLV, albeit at a level less than that with wild-type tRNAPhe. Collectively, our results demonstrate that the tRNA primer captured by HIV-1 and MuLV occurs after nuclear export of tRNA and supports a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis.

scholarly journals Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

1981 ◽  
Vol 1 (10) ◽  
pp. 902-909 ◽  
Author(s):  
C B Hirschberg ◽  
R M Baker ◽  
M Perez ◽  
L A Spencer ◽  
D Watson
Keyword(s):  
Chinese Hamster Ovary ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Wild Type ◽  
Ovary Cells ◽  
Replica Plating ◽  
Selection Of

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.

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