PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 9491397

The International Comet Assay Workshop.

D Anderson·M J Plewa
View original source ↗Export metadata ↓

Abstract

The Comet (single cell gel electrophoresis) assay primarily measures DNA strand breakage in single cells (Singh et al., 1988). Briefly, cells are suspended in low melting point agarose on a microscope slide. The slides are put in lysing buffer to allow the DNA to unwind and then in electrophoresis buffer. During electrophoresis the broken DNA moves towards the anode forming a Comet tail, with the greater the extent of damage, the greater the tail. Assays can be conducted under neutral or alkaline (> pH 13) conditions. Double-strand breaks are measured under neutral conditions and single-strand breaks under alkaline conditions, where abasic sites and other alkali-labile sites or intermediates in base or nucleotide excision repair can also be detected. There are several good reviews concerning the assay (McKelvey-Martin et al., 1993; Fairbairn et al., 1995; Tice, 1995).

Explore related subjects

Cell ExtractsCell LineClinical ProtocolsDNA DamageDNA RepairElectrophoresis, Agar GelGerm CellsHumansHybrid CellsConference ProceedingsResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

D Anderson, M J Plewa. 1998. The International Comet Assay Workshop.. https://doi.org/10.1093/mutage%2F13.1.67

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

A method for the effective depletion of albumin from cellular extracts: application to human myocardium.

Proteomic analysis of large numbers of proteins is assisted if each protein species is present at approximately equal concentrations. As such, the extraction of proteins from tissue samples should be designed to maintain a limited dynamic range in the concentration of proteins present. However, in many tissue extracts a high concentration of serum albumin exists from tissue perfusion and/or an inability to effectively rinse the tissue owing to surgical limitations. The analysis of these tissues would be assisted if contaminating serum albumin could be reduced. This chapter outlines a protocol for the effective reduction of serum albumin levels from human myocardium extracts enriched for soluble cytoplasmic proteins.

Cell Extracts↗
PubMed2006

The presence of a common downstream box enables the simultaneous expression of multiple proteins in an E. coli extract.

In our experiments to produce different combinations of recombinant proteins in a cell-free protein synthesis system derived from Escherichia coli, we found that certain pairs of ORFs were not expressed equally. Instead, only a single DNA species was expressed dominantly, while the expression of the others was almost completely repressed. This bias during the co-expression of the DNA pairs was eliminated when an identical downstream box sequence was added to the 5'-ends of the template DNA pairs. By introducing identical nucleotide sequences of the his-tag or the downstream box of chloramphenicol acetyltransferase (CAT-DB) in front of the target genes that were otherwise not expressed compatibly, both of the encoded proteins were produced at similar productivities. Moreover, in the presence of a common downstream box, multiple genes were simultaneously expressed in the same reaction mixture. We expect that the proposed approach will offer a powerful tool for the preparation of unbiased protein libraries, as well as for studying the structure and functions of interacting proteins.

Cell Extracts↗
PubMed2006

Neutrophil elastase, an innate immunity effector molecule, represses flagellin transcription in Pseudomonas aeruginosa.

Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors triggers an innate immune response to colonizing or invading bacteria. Conversely, many bacteria have evolved mechanisms to dampen this response by downregulating the synthesis of such PAMPs. We have previously demonstrated that Pseudomonas aeruginosa growing in mucopurulent human respiratory mucus from cystic fibrosis patients represses the expression of its flagellin, a potent stimulant of the innate immune response. Here we demonstrate that this phenomenon occurs in response to the presence of neutrophil elastase in such mucus. Nonpurulent mucus from animals had no such repressive effect. Furthermore, lysed neutrophils from human blood reproduced the flagellin-repressive effect ex mucus and, significantly, had no effect on the viability of this organism. Neutrophil elastase, a component of the innate host defense system, has been described to be bactericidal for gram-negative bacteria and to degrade bacterial virulence factors. Thus, the resistance of P. aeruginosa to the bactericidal effect of neutrophil elastase, as well as this organism's ability to sense this enzyme's presence and downregulate the synthesis of a PAMP, may be the key factors in allowing P. aeruginosa to colonize the lungs. These findings demonstrate the dynamic nature of this bacterium's response to host defenses that ensures its success as a colonizer and also highlights the dual nature of defense molecules that confer advantages and disadvantages to both hosts and pathogens.

Cell Extracts↗