A novel method for assessment of transcription activation in vitro for bacterial promoter

69-73In vitro transcription (IVT) assay is a useful tool to monitor the transcriptional activity of a specific promoter under defined conditions in vitro. Conventional IVT assay involves use of radiolabeled probes which makes it tedious to perform and limits its utility for large scale applications. Here, a reverse-transcription-PCR (RT-PCR) based transcript detection method has been developed for bacterial in vitro transcription assay. Unlike conventional radiolabeling approach, this method is simple, fast, needs smaller reaction volumes, does not require special infrastructure and could be potentially used for any large-scale screening applications. The present study demonstrates the feasibility of this method by showing Cyclic AMP Receptor Protein (CRP) mediated activation of a CRP dependent E. coli promoter

A novel method for assessment of in vitro transcription for a bacterial promoter

In vitro transcription (IVT) assay is a useful tool to monitor the transcriptional activity of a specific promoter under defined conditions in vitro. Conventional IVT assay involves use of radiolabeled probes which makes it tedious to perform and limits its utility for large scale applications. Here, a reverse-transcription-PCR (RT-PCR) based transcript detection method has been developed for bacterial in vitro transcription assay. Unlike conventional radiolabeling approach, this method is simple, fast, needs smaller reaction volumes, does not require special infrastructure and could be potentially used for any large-scale screening applications. The present study demonstrates the feasibility of this method by showing Cyclic AMP Receptor Protein (CRP) mediated activation of a CRP dependent E. coli promoter

Assessment of hybridization among wild and cultivated Vigna unguiculata subspecies revealed by arbitrarily primed polymerase chain reaction analysis

This paper show that inter-subspecies hybridization among certain Vigna unguiculata subspecies, occurred during the course of evolution. This has affected several regions of the genome and is interfering with the dependable assessment of sub-species relationships using single (rRNA regions) or multilocus markers

Loss of Expression of cspC, a Cold Shock Family Gene, Confers a Gain of Fitness in Escherichia coli K-12 Strains

The CspA family of cold shock genes in Escherichia coli K-12 includes nine paralogs, cspA to cspI. Some of them have been implicated in cold stress adaptation. Screening for mutations among common laboratory E. coli strains showed a high degree of genetic diversity in cspC but not in cspA and cspE. This diversity in cspC was due to a wide spectrum of variations including insertions of IS elements, deletion, and point mutation. Northern analysis of these mutants showed loss of cspC expression in all but one case. Further analysis of the loss-of-function cspC mutants showed that they have a fitness advantage in broth culture after 24 h over their isogenic wild-type derivatives. Conversely, introduction of mutated cspC alleles conferred a competitive fitness advantage to AB1157, a commonly used laboratory strain. This provides the evidence that loss of cspC expression is both necessary and sufficient to confer a gain of fitness as seen in broth culture over 24 h. Together, these results ascribe a novel role in cellular growth at 37°C for CspC, a member of the cold shock domain-containing protein family

Effects of Glutathione and Ascorbic Acid on Streptomycin Sensitivity of Escherichia coli

We examined the effects of antioxidants and the role of reactive oxygen species (ROS) on the antibacterial action of aminoglycosides in Escherichia coli. We concluded that reduced streptomycin sensitivity in the presence of glutathione and ascorbic acid is not due to the antioxidant-mediated scavenging of ROS

Evaluation of long primers for AP-PCR analysis of mungbean [<i style="">Vigna radiata</i> (L.) Wilczek]: Genetic relationships and fingerprinting of some genotypes

511-518There are a number of applications of molecular markers in agriculture such as assessing genetic diversity, generating DNA fingerprints and developing markers linked to a trait of interest, etc. Arbitrarily Primed-Polymerase Chain Reaction (AP-PCR) in which template DNA is amplified using single arbitrary primers of 10 base in length is a widely used technique. The objectives of this investigation were: (a) to compare efficiency of long primers (ranging from 18 to 22 base in length) and 10 base primers in detecting RAPDs in mungbean and (b) to evaluate some selected long primers for their ability to discriminate 46 mungbean genotypes and to study the genetic relationships among them. Both the groups of primers were evaluated for the total number of discrete and detectable amplified fragments and polymorphic bands detected between two mungbean genotypes. The long primers yielded significantly higher number of discrete and detectable bands as well as polymorphic bands than 10 base primers. A set of eight long primers was used for AP-PCR analysis of 46 mungbean genotypes. A total of 173 fragments were amplified of which 39.08 % were polymorphic. AP-PCR profiles from only three primers were sufficient to differentiate all the genotypes. A high degree of genetic variation was observed among different genotypes, whereas, those originating from the same source were highly related. The results show that long primers can be used for efficiently analysing genetic diversity and the relationships in a large mungbean germplasm collection

Resveratrol induced inhibition of Escherichia coli proceeds via membrane oxidation and independent of diffusible reactive oxygen species generation

Resveratrol (5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol), a redox active phytoalexin with a large number of beneficial activities is also known for antibacterial property. However the mechanism of action of resveratrol against bacteria remains unknown. Due to its extensive redox property it was envisaged if reactive oxygen species (ROS) generation by resveratrol could be a reason behind its antibacterial activity. Employing Escherichia coli as a model organism we have evaluated the role of diffusible reactive oxygen species in the events leading to inhibition of this organism by resveratrol. Evidence for the role of ROS in E. coli treated with resveratrol was investigated by direct quantification of ROS by flow cytometry, supplementation with ROS scavengers, depletion of intracellular glutathione, employing mutants devoid of enzymatic antioxidant defences, induction of adaptive response prior to resveratrol challenge and monitoring oxidative stress response elements oxyR, soxS and soxR upon resveratrol treatment. Resveratrol treatment did not result in scavengable ROS generation in E. coli cells. However, evidence towards membrane damage was obtained by potassium leakage (atomic absorption spectrometry) and propidium iodide uptake (flow cytometry and microscopy) as an early event. Based on the comprehensive evidences this study concludes for the first time the antibacterial property of resveratrol against E. coli does not progress via the diffusible ROS but is mediated by site-specific oxidative damage to the cell membrane as the primary event

Hydroxylamine inhibition of the nitrate reductase complex from Amaranthus

Nitrate reductase from Amaranthus viridis is similar to nitrate reductase from other plant sources. NH<SUB>2</SUB>OH inhibits nitrate reduction from NADH by the nitrate reductase complex, but it does not inhibit either the NADH-dehydrogenase activity or nitrate reduction from reduced flavin mononucleotides. The inhibition observed was non-competitive with nitrate when the enzyme was pre-incubated with NH<SUB>2</SUB>OH and NADH, and competitive with nitrate without pre-incubation. The K<SUB>i</SUB> values for NH<SUB>2</SUB>OH were 5 &#956;M and 30 &#956;M with or without pre-incubation respectively