A forward mutation assay using ampicillin-resistance in Escherichia coli designed for investigating the mutagenicity of biological samples

Mutagenesis ◽  
1987 ◽  
Vol 2 (6) ◽  
pp. 455-467 ◽  
Author(s):  
David Bosworth ◽  
Christopher Crofton-Sleigh ◽  
Stanley Venitt
Keyword(s):  
Escherichia Coli ◽  
Biological Samples ◽  
Ampicillin Resistance ◽  
Mutation Assay ◽  
Forward Mutation

Large-scale production of citrate synthase from a cloned gene

1982 ◽  
Vol 60 (12) ◽  
pp. 1143-1147 ◽  
Author(s):  
Harry W. Duckworth ◽  
Alexander W. Bell
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Citrate Synthase ◽  
Specific Activity ◽  
Tetracycline Resistance ◽  
Scale Production ◽  
Ampicillin Resistance ◽  
Colicin E1 ◽  
Large Scale Production ◽  
Cloned Gene

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

A novel bacterial reversion and forward mutation assay based on green fluorescent protein

1998 ◽  
Vol 414 (1-3) ◽  
pp. 95-105 ◽  
Author(s):  
Neal F Cariello ◽  
Sabrina Narayanan ◽  
Puntipa Kwanyuen ◽  
Heidi Muth ◽  
Warren M Casey
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
Mutation Assay ◽  
Forward Mutation

scholarly journals The TREX2 3′→ 5′ Exonuclease Physically Interacts with DNA Polymerase δ and Increases Its Accuracy

2002 ◽  
Vol 2 ◽  
pp. 275-281 ◽  
Author(s):  
Igor V. Shevelev ◽  
Kristijan Ramadan ◽  
Ulrich Hubscher
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Error Rates ◽  
High Fidelity ◽  
Replication Machinery ◽  
Dna Polymerase Δ ◽  
Pol I ◽  
Calf Thymus ◽  
Mutation Assay ◽  
Forward Mutation

Proofreading function by the 3′→ 5′ exonuclease of DNA polymerase δ (pol δ) is consistent with the observation that deficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol δdfrom isotonic (pol δi) and detergent (pol δd) calf thymus extracts. Pol δdhad a 20-fold higher ratio of exonuclease to DNA polymerase than pol δi. This was due to the physical association of the TREX2 exonuclease to pol δd, which was missing from pol δi. Pol δdwas fivefold more accurate than pol δiunder error-prone conditions (1 μM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 μM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3′→ 5′ exonucleases, such as TREX2, through its association with pol I can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.

Responses of the L5178Y tk+/tk− mouse lymphoma cell forward mutation assay ii: 18 coded chemicals

1988 ◽  
Vol 11 (1) ◽  
pp. 91-118 ◽  
Author(s):  
Douglas B. McGregor ◽  
Alison Brown ◽  
Pamela Cattanach ◽  
Ian Edwards ◽  
Douglas McBride ◽  
...  
Keyword(s):  
Lymphoma Cell ◽  
Mutation Assay ◽  
Forward Mutation ◽  
Mouse Lymphoma

scholarly journals Recovery of Plasmid pEMB1, Whose Toxin-Antitoxin System Stabilizes an Ampicillin Resistance-Conferring β-Lactamase Gene in Escherichia coli, from Natural Environments

2014 ◽  
Vol 81 (1) ◽  
pp. 40-47 ◽  
Author(s):  
Hyo Jung Lee ◽  
Hyun Mi Jin ◽  
Moon Su Park ◽  
Woojun Park ◽  
Eugene L. Madsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Stability ◽  
Functional Characterization ◽  
Treatment Plant ◽  
Antibiotic Resistance Genes ◽  
Cloning And Expression ◽  
Natural Environments ◽  
Ampicillin Resistance ◽  
Content Type ◽  
Lactamase Gene

ABSTRACTNon-culture-based procedures were used to investigate plasmids showing ampicillin resistance properties in two different environments: remote mountain soil (Mt. Jeombong) and sludge (Tancheon wastewater treatment plant). Total DNA extracted from the environmental samples was directly transformed intoEscherichia coliTOP10, and a single and three different plasmids were obtained from the mountain soil and sludge samples, respectively. Interestingly, the restriction fragment length polymorphism pattern of the plasmid from the mountain soil sample, designated pEMB1, was identical to the pattern of one of the three plasmids from the sludge sample. Complete DNA sequencing of plasmid pEMB1 (8,744 bp) showed the presence of six open reading frames, including a β-lactamase gene. Using BLASTX, theorf5andorf6genes were suggested to encode a CopG family transcriptional regulator and a plasmid stabilization system, respectively. Functional characterization of these genes using a knockoutorf5plasmid (pEMB1ΔparD) and the cloning and expression oforf6(pET21bparE) indicated that these genes were antitoxin (parD) and toxin (parE) genes. Plasmid stability tests using pEMB1 and pEMB1ΔparDEinE. colirevealed that theorf5andorf6genes enhanced plasmid maintenance in the absence of ampicillin. Using a PCR-based survey, pEMB1-like plasmids were additionally detected in samples from other human-impacted sites (sludge samples) and two other remote mountain soil samples, suggesting that plasmids harboring a β-lactamase gene with a ParD-ParE toxin-antitoxin system occurs broadly in the environment. This study extends knowledge about the dissemination and persistence of antibiotic resistance genes in naturally occurring microbial populations.

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