scholarly journals Reversion of argE3 to Arg(+) in Escherichia coli AB1157 -an informative bacterial system for mutation detection.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Anna Sikora ◽  
Elżbieta Grzesiuk
Keyword(s):  
Dna Damage ◽  
Mutation Detection ◽  
Detection System ◽  
Base Substitution ◽  
E Coli ◽  
Level Ii ◽  
Nonsense Codon ◽  
Amber Suppressor ◽  
Detection Of Mutations

This review concerns reversion of the argE3 (ochre) nonsense mutation to prototrophy in E. coli AB1157 strain as an informative system for mutation detection. Strain AB1157 bears the argE3 (ochre), hisG4 (ochre) and thr-1 (amber) mutations, and the supE44 amber suppressor on its chromosome. The Arg(+) phenotype can be restored by (i) any base substitution at the argE3 site that changes the nonsense UAA codon to any sense nucleotide triplet or to UAG recognized by the supE44 amber suppressor, or (ii) suppressor mutations enabling the reading of the UAA nonsense codon. The argE3 → Arg(+) reversion-based system enables (i) determination of the spontaneous or induced mutation level; (ii) determination of base substitutions (suppressor analysis); (iii) examination of transcription-coupled repair (TCR) since targets for DNA damage are situated on the transcribed or coding strand of DNA; (iv) detection of mutations resulting from single stranded DNA damage. This review focuses on studies carried out since the early 1990s till now with the application of the AB1157-based mutation detection system. Recently, the system has been used to obtain new data on the processes of methyl methanesulfonate-induced mutagenesis and DNA repair in E. coli alkB⁻ mutants.

scholarly journals Genotypic Determination of Mycobacterium tuberculosisAntibiotic Resistance Using a Novel Mutation Detection Method, the Branch Migration Inhibition M. tuberculosis Antibiotic Resistance Test

2000 ◽  
Vol 38 (10) ◽  
pp. 3656-3662 ◽  
Author(s):  
Y. P. Liu ◽  
M. A. Behr ◽  
P. M. Small ◽  
N. Kurn
Keyword(s):  
Nucleic Acids ◽  
Genomic Dna ◽  
Mutation Detection ◽  
Detection Method ◽  
Clinical Isolates ◽  
Base Substitution ◽  
Enzyme Linked Immunosorbent Assay ◽  
Branch Migration ◽  
Migration Inhibition

A novel method for the detection of any alteration within a defined sequence has recently been demonstrated (A. Lishanski, N. Kurn, and E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin. Chem. 46:9, 2000). Essential to this method are the generation of partial duplexes that are capable of forming four-stranded structures and the ability to detect inhibition of branch migration in these structures (I. G. Panyutin and P. Hsieh, J. Mol. Biol. 230:413–424, 1993). Inhibition of branch migration indicates the presence of sequence alteration. This mutation detection method, termed branch migration inhibition (BMI), is suitable for the detection of drug resistance in M. tuberculosis, which is frequently associated with multiple mutations within known genes. We describe the genotypic determination of the rifampin (RMP) and pyrazinamide (PZA) susceptibilities of M. tuberculosis isolates, using BMI coupled with the luminescence oxygen channeling immunoassay (LOCI) (E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426–5430, 1994). RMP and PZA resistances are associated with multiple mutations within the rpoB and pncA genes, respectively.M. tuberculosis genomic DNA samples prepared from 46 clinical isolates were used for genotypic determination of RMP resistance in a “blind study.” Similarly, PZA resistance was determined using genomic DNA samples prepared from 37 clinical isolates. Full agreement of the genotypic and phenotypic determinations of drug susceptibility was demonstrated. RMP susceptibility determination directly from cells of 10 clinical isolates grown in culture was also demonstrated. The genotypic result of only 1 out of 10 isolates did not agree with the phenotypic susceptibility testing result. Sequence analysis of the rpoB gene of this clinical isolate revealed a single base substitution, most likely a silent point mutation. The new BMI-LOCI mutation detection method is a rapid and accurate procedure for the genotypic determination of the RMP and PZA susceptibilities of M. tuberculosis clinical isolates. BMI can also be detected by using commercially available automated enzyme-linked immunosorbent assay plate formats (Lishanski et al., Nucleic Acids Res. 28:E42, 2000).

Concentration determination of chromatin in unstained resin-embedded sections by means of Z-contrast in STEM

1990 ◽  
Vol 48 (2) ◽  
pp. 186-187
Author(s):  
M. Haider ◽  
B. Bohrmann
Keyword(s):  
Energy Loss ◽  
Freeze Substitution ◽  
Biological Macromolecules ◽  
Local Concentration ◽  
E Coli ◽  
Concentration Determination ◽  
Phosphorous Content ◽  
Z Contrast ◽  
Electron Energy Loss

The technique of Z-contrast in STEM offers the possibility to determine the local concentration of macromolecules like lipids, proteins or DNA. Contrast formation depends on the atomic composition of the particular structure. In the case of DNA, its phosphorous content discriminates it from other biological macromolecules. In our studies, sections of E. coli, the dinoflagellate Amphidinium carterae and Euglena spec. cells were used which were obtained by cryofixation followed by freeze-substitution into acetone with 3% glutaraldehyde. The samples were then embedded either in Lowicryl HM20 at low temperature or in Epon at high temperature. Sections were coated on both sides with 30Å carbon.The DF- and the inelastic image have been recorded simultaneously with a Cryo-STEM. This Cryo-STEM is equipped with a highly dispersive Electron Energy Loss Spectrometer. With this instrument pure Z-contrast can be achieved either with a Filtered DF-image divided by the inelastic image or, as is used in this paper, by dividing the conventional DF-image by an inelastic image which has been recorded with an inelastic detector whose response is dependent on the total energy loss of the inelastically scattered electrons.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

2019 ◽  
Vol 22 (5) ◽  
pp. 346-354
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Large Scale ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Reporter System ◽  
E Coli ◽  
Starting Point ◽  
High Concentrations ◽  
Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

Novel sliding hybrid microchip detection system for determination of whole blood phosphorus concentration

2021 ◽  
Vol 419 ◽  
pp. 129592
Author(s):  
Chin-Chung Tseng ◽  
Szu-Jui Chen ◽  
Song-Yu Lu ◽  
Chien-Hsuan Ko ◽  
Ju-Ming Wang ◽  
...  
Keyword(s):  
Whole Blood ◽  
Detection System ◽  
Phosphorus Concentration

scholarly journals ANTIMICROBIAL AND ANTIOXIDANT EFFECT OF NATURAL EXTRACTS FROM LEAVES, ROOT, STEM AND FLOWERS OF BACCHARIS LATIFOLIA FROM ECUADOR

2020 ◽  
pp. 78-84
Author(s):  
FAVIAN BAYAS-MOREJON ◽  
ANGELICA TIGRE ◽  
RIVELINO RAMON ◽  
DANILO YANEZ
Keyword(s):  
Antimicrobial Activity ◽  
Antioxidant Activity ◽  
Potassium Phosphate ◽  
Natural Antioxidants ◽  
Butylated Hydroxytoluene ◽  
Penicillin G ◽  
Diffusion Method ◽  
E Coli ◽  
Antibacterial And Antioxidant Activity

Objective: The increase in chronic and degenerative diseases and the use of synthetic antioxidants such as (butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT)) are being restricted because they can be considered carcinogenic. Therefore, there is a growing interest in the search for natural antioxidants, especially from plants, due to their content in different bioactive compounds, such as antioxidants and antimicrobials. To evaluate the antibacterial and antioxidant activity of Baccharislatifolia extracts. Methods: For the determination of the antimicrobial activity of extracts of leaves, root, stem and flowers of Baccharislatifolia (Bl), the disk plate diffusion method was used, the strains of Listeria, Salmonella and E. coli were studied; antibiotics Penicillin G and Ciprofloxacin were the controls. For the antioxidant activity, a solution of H2O2 (Abs at 230 nm) was prepared in Potassium Phosphate Monobasic-Sodium Hydroxide buffer. Results: The antimicrobial activity against Listeria and Salmonella, showed that the extracts of leaves and flowers were more effective with inhibition zones>15 mm and>20 mm respectively. In front of E. coli, the extracts of flowers and stem were the best with zones>7.0 mm. Antibiotics studied inhibited the development of Listeria and Salmonella. However, E. coli isolates were resistant. In the antioxidant activity, the flower extract of Bl in 60 mg/ml presents a higher effect with 47.25%. Conclusion: Bl extracts from leaves and flowers were more efficient both in their antimicrobial and antioxidant capacity.

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