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.

scholarly journals Mutations Reducing In Vitro Susceptibility to Novel LpxC Inhibitors in Pseudomonas aeruginosa and Interplay of Efflux and Nonefflux Mechanisms

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Adriana K. Jones ◽  
Ruth E. Caughlan ◽  
Angela L. Woods ◽  
Kyoko Uehara ◽  
Lili Xie ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Single Step ◽  
Protein Overexpression ◽  
Wild Type ◽  
Novel Mutations ◽  
In Vitro Susceptibility ◽  
Content Type ◽  
Selection Experiments ◽  
Step Selection

ABSTRACT Upregulated expression of efflux pumps, lpxC target mutations, LpxC protein overexpression, and mutations in fabG were previously shown to mediate single-step resistance to the LpxC inhibitor CHIR-090 in P. aeruginosa. Single-step selection experiments using three recently described LpxC inhibitors (compounds 2, 3, and 4) and mutant characterization showed that these mechanisms affect susceptibility to additional novel LpxC inhibitors. Serial passaging of P. aeruginosa wild-type and efflux pump-defective strains using the LpxC inhibitor CHIR-090 or compound 1 generated substantial shifts in susceptibility and underscored the interplay of efflux and nonefflux mechanisms. Whole-genome sequencing of CHIR-090 passage mutants identified efflux pump overexpression, fabG mutations, and novel mutations in fabF1 and in PA4465 as determinants of reduced susceptibility. Two new lpxC mutations, encoding A214V and G208S, that reduce susceptibility to certain LpxC inhibitors were identified in these studies, and we show that these and other target mutations differentially affect different LpxC inhibitor scaffolds. Lastly, the combination of target alteration (LpxCA214V) and upregulated expression of LpxC was shown to be tolerated in P. aeruginosa and could mediate significant decreases in susceptibility.

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 GALACTOKINASE MUTANTS OF CHINESE HAMSTER SOMATIC CELLS RESISTANT TO 2-DEOXYGALACTOSE

Genetics ◽  
1976 ◽  
Vol 83 (1) ◽  
pp. 137-147
Author(s):  
Jean-Paul Thirion ◽  
Denis Banville ◽  
Henri Noel
Keyword(s):  
Diagnostic Test ◽  
Somatic Cells ◽  
Chinese Hamster ◽  
Single Step ◽  
The Other ◽  
Kinetic Properties ◽  
Wild Type ◽  
Structural Mutation ◽  
Step Selection

ABSTRACT The growth of Chinese hamster somatic cells was inhibited by 0.2 mg/cc of 2-deoxygalactose. Mutants partially or fully resistant to 2-deoxygalactose were isolated in a single-step or two-step selection. Some of them did not grow as well as the wild type; one of them which lacked galactokinase (EC.2.7.1.6) activity did not grow at all in galactose medium. The galactokinase kinetic properties (Vmax & Kmax) of the other mutants and of the wild type were different. Therefore resistance resulted either from the possible absence of galactokinase synthesis or from a structural mutation, possibly a missence mutation, in the galactokinase gene.—A simple diagnostic test for juvenile cataract is proposed.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles

1983 ◽  
Vol 3 (7) ◽  
pp. 1172-1181
Author(s):  
W E Bradley
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
High Frequency ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Class Ii ◽  
Class I ◽  
Wild Type ◽  
Ovary Cells

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.

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.

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.

scholarly journals Synthetic chimeric nucleases function for efficient genome editing

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
R. M. Liu ◽  
L. L. Liang ◽  
E. Freed ◽  
H. Chang ◽  
E. Oh ◽  
...  
Keyword(s):  
Kinetic Parameter ◽  
Genome Editing ◽  
Cell Lines ◽  
Human Cell ◽  
Human Cell Lines ◽  
Wild Type ◽  
Functional Variants ◽  
Protospacer Adjacent Motif ◽  
Natural Variants ◽  
Selection Of

AbstractCRISPR–Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demonstration opens up the possibility of generating nuclease sequences with implications across biotechnology.

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