Transfected DNA is mutated in monkey, mouse, and human cells.

Papovavirus-based shuttle vectors containing the bacterial lacI gene were used to show that a mutation frequency in the range of 1% occurs in lacI when such vectors are transfected into COS7 and CV-1 simian cells, NIH 3T3, 3T6, L, and C127 mouse cells, and human 293 and HeLa cells. This frequency is approximately four orders of magnitude higher than the spontaneous mutation frequency in either mammalian or bacterial cells. The mutations are predominantly base substitutions and deletions and also include insertions from the mammalian genome. Time course experiments argue that mutagenesis occurs soon after arrival of the DNA into the nucleus. However, replication of the vector is not required since mutations occur even when the vector lacks all viral sequences. The high mutation frequency appears to be the characteristic outcome of transfection of DNA into mammalian cells.

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SPONTANEOUS UNSTABLE UNC-22 IV MUTATIONS IN C. ELEGANS VAR. BERGERAC

Genetics ◽

10.1093/genetics/108.4.859 ◽

1984 ◽

Vol 108 (4) ◽

pp. 859-877

Author(s):

D G Moerman ◽

R H Waterston

Keyword(s):

Mutation Frequency ◽

Spontaneous Mutation ◽

Phenotypic Effect ◽

C Elegans ◽

High Mutation Frequency ◽

Mutator Activity ◽

Discrete Site ◽

Unstable Mutations ◽

Spontaneous Mutation Frequency ◽

Forward Mutation

ABSTRACT This paper describes a mutator system in the nematode Caenorhabditis elegans var. Bergerac for the gene unc-22. Of nine C. elegans and two C. briggsae strains tested only the Bergerac BO strain yielded mutant animals at a high frequency and the unc-22 IV gene is a preferred mutational target. The forward spontaneous mutation frequency at the unc-22 locus in Bergerac BO is about 1 × 10-4, and most of these spontaneous unc-22 mutations revert at frequencies between 2 × 10-3 and 2 × 10-4. Both the forward mutation frequency and the reversion frequency are sensitive to genetic background. Spontaneous unc-22 mutations derived in a Bergerac background and placed in a primarily Bristol background revert at frequencies of <10-6. When reintroduced into a Bergerac/Bristol hybrid background the mutations once again become unstable. The mutator activity could not be localized to a discrete site in the Bergerac genome. Nor did mutator activity require the Bergerac unc-22 gene as a target since the Bristol unc-22 homolog placed in a Bergerac background also showed high mutation frequency. Intragenic mapping of two spontaneous unc-22 alleles, st136 and st137, place both mutations in the central region of the known unc-22 map. However, these mutations probably recombine with one another, suggesting that the unstable mutations can occur in more than one site in unc-22. Examination of the phenotypic effect of these mutations on muscle structure indicates that they are less severe in their effect than a known amber allele. We suggest that this mutator system is polygenic and dispersed over the nematode genome and could represent activity of the transposable element Tc1.

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In Vitro Antifungal Activity and Cytotoxicity of a Novel Membrane-Active Peptide

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.7.1704 ◽

1999 ◽

Vol 43 (7) ◽

pp. 1704-1707 ◽

Cited By ~ 17

Author(s):

Sung Yu Hong ◽

Jong Eun Oh ◽

Keun Hyeung Lee

Keyword(s):

Antifungal Activity ◽

Mammalian Cells ◽

Time Course ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Jurkat Cells ◽

Active Peptide ◽

Fluconazole Resistant ◽

Nih 3T3

ABSTRACT In the present study, we investigated the antifungal activity and cytotoxicity of a novel membrane-active peptide, KKVVFKVKFKK (MP). MP inhibited the growth of various pathogenic fungi isolated from patients and of fluconazole-resistant fungi at concentrations of 2 to 32 μg/ml. MP had potent fungicidal activity; the minimal fungicidal concentrations of the peptide were no more than fourfold greater than the MICs. Time course experiments of MP-induced killing of Candida albicans ATCC 36232 showed that the rate of killing was rapid and depended on the concentration of MP. MP had a strong synergism with other antifungal drugs; the fractional inhibitory concentration index values of MP with amphotericin B and fluconazole for C. albicans were 0.16 and 0.02, respectively. The 50% inhibitory concentrations of MP for NIH 3T3 and Jurkat cells were approximately 100 times higher than the MIC for C. albicansATCC 36232, indicating that MP had high selectivity between the fungal and mammalian cells. These results suggest that MP has great advantages in the development of antifungal agents.

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High mutation frequency in DNA transfected into mammalian cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.80.10.3015 ◽

1983 ◽

Vol 80 (10) ◽

pp. 3015-3019 ◽

Cited By ~ 114

Author(s):

M. P. Calos ◽

J. S. Lebkowski ◽

M. R. Botchan

Keyword(s):

Mammalian Cells ◽

Mutation Frequency ◽

High Mutation Frequency

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Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis

Biomolecules ◽

10.3390/biom11060843 ◽

2021 ◽

Vol 11 (6) ◽

pp. 843

Author(s):

Balagra Kasim Sumabe ◽

Synnøve Brandt Ræder ◽

Lisa Marie Røst ◽

Animesh Sharma ◽

Eric S. Donkor ◽

...

Keyword(s):

Mammalian Cells ◽

Mutation Frequency ◽

Nucleoside Analogues ◽

Cancer Therapies ◽

E Coli ◽

Replicative Stress ◽

Pol V ◽

Dna And Rna ◽

Sos Induction ◽

Anti Cancer

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

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Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.7.2698 ◽

1988 ◽

Vol 8 (7) ◽

pp. 2698-2704 ◽

Cited By ~ 36

Author(s):

I W Caras ◽

D W Martin

Keyword(s):

Ribonucleotide Reductase ◽

Mammalian Cells ◽

Mutant Mouse ◽

Chinese Hamster Ovary Cells ◽

Spontaneous Mutation ◽

Single Point ◽

Chinese Hamster ◽

Fold Increase ◽

Single Point Mutation ◽

Mutator Phenotype

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.

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Determination of the spontaneous mutation frequency per cell per generation in human diploid fibroblasts cultured at different temperatures

Mutation Research/Environmental Mutagenesis and Related Subjects ◽

10.1016/0165-1161(83)90153-x ◽

1983 ◽

Vol 113 (3-4) ◽

pp. 306

Author(s):

J.W.I.M. Simons

Keyword(s):

Mutation Frequency ◽

Spontaneous Mutation ◽

Diploid Fibroblasts ◽

Human Diploid Fibroblasts ◽

Different Temperatures ◽

Spontaneous Mutation Frequency

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Modulation of the transport of a lysosomal enzyme by PDGF.

The Journal of Cell Biology ◽

10.1083/jcb.110.2.319 ◽

1990 ◽

Vol 110 (2) ◽

pp. 319-326 ◽

Cited By ~ 46

Author(s):

E M Prence ◽

J M Dong ◽

G G Sahagian

Keyword(s):

Secretory Pathway ◽

Cathepsin L ◽

Control Level ◽

3T3 Cells ◽

Lysosomal Protease ◽

Affinity Ligand ◽

The Media ◽

Mouse Cells ◽

Nih 3T3 ◽

The Relationship

The major excreted protein (MEP) of transformed mouse fibroblasts is the lysosomal protease, cathepsin L. MEP is also secreted by untransformed mouse cells in response to growth factors and tumor promoters, and is thought to play a role in cell growth and transformation. To determine the relationship between MEP synthesis and MEP secretion, we have examined these events in PDGF-treated NIH 3T3 cells. PDGF enhanced MEP synthesis and caused the diversion of MEP from the lysosomal delivery pathway to a secretory pathway. These two effects were found to be regulated independently at various times after growth factor addition. Short PDGF treatments (0.5 or 1 h) resulted in quantitative secretion of MEP although synthesis was near the control level. High levels of both synthesis and secretion occurred between 2 and 14 h of PDGF treatment. Between 18 and 30 h, the amount of secreted MEP returned to the low control level even though synthesis remained elevated. The secretion was specific for MEP; other lysosomal enzymes were not found in the media from PDGF-treated cells. PDGF-induced secretion of MEP was inhibited 84% by cycloheximide, suggesting that protein synthesis is required to elicit this effect. PDGF also caused a time-dependent increase in mannose 6-phosphate (Man-6-P) receptor-mediated endocytosis. These data support a model in which PDGF alters the distribution of Man-6-P receptors such that the Golgi concentration of receptors becomes limiting, thereby causing the selective secretion of the low affinity ligand, MEP.

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Expression of Heterologous Cellulases inThermotogasp. Strain RQ2

BioMed Research International ◽

10.1155/2015/304523 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 8

Author(s):

Hui Xu ◽

Dongmei Han ◽

Zhaohui Xu

Keyword(s):

Enzyme Activities ◽

Bacterial Cells ◽

Recombinant Enzymes ◽

Caldicellulosiruptor Saccharolyticus ◽

Shuttle Vectors ◽

E Coli ◽

Chimeric Enzymes ◽

Coding Regions ◽

First Time ◽

Culture Supernatants

The ability ofThermotogaspp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) fromCaldicellulosiruptor saccharolyticuswere cloned intoT.sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried byThermotoga-E. colishuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed inE. coliDH5αandT.sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. InE. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas inT.sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost inT.sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed inThermotoga, demonstrating the feasibility of using engineeredThermotogaspp. for efficient cellulose utilization.

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The Use of Bacteriophage for Detection and Biocontrol of Foodborne Pathogen

Indonesian Bulletin of Animal and Veterinary Sciences ◽

10.14334/wartazoa.v28i1.1791 ◽

2018 ◽

Vol 28 (1) ◽

pp. 33

Author(s):

Tati Ariyanti

Keyword(s):

Mammalian Cells ◽

Food Industry ◽

Pathogenic Bacteria ◽

Foodborne Pathogen ◽

Foodborne Disease ◽

Bacterial Cells ◽

Detection Tool ◽

Specific Receptors ◽

Antibiotics Detection ◽

Potential Use

Bacteriophages are viruses that have ability to attack bacterial cells in specific receptors, infect, multiply in bacterial cells and eventually lyse bacterial cells. This unique bacteriophage character is highly beneficial because it is harmless to mammalian cells and does not interfere with natural microbes. Bacteriophages are easy to obtain because they are widespread in the environment such as soil, water, animal, and farm waste or food. This paper describes the potential use of bacteriophages to detect pathogen and foodborne pathogen biocontrol. Bacteriophages are very potential to control the growth of pathogenic bacteria both in food industry and environment. Bacteriophages act as antibiotics, detection tool for pathogenic bacteria in the food chain, food biopreservative from pathogen bacteria contamination, and foodborne disease prevention. Although research on bacteriophage in Indonesia has not been widely reported, research on bacteriophage utilization is being carried on.

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