Affinity purification of plasmid DNA by temperature-triggered precipitation

This protocol presents a new method to purify plasmid DNA using temperature-triggered precipitation. The principle is based on the specific DNA-binding affinity of a bacterial metalloregulatory (MerR) protein to its cognate DNA sequence and the temperature responsiveness of elastin-like protein (ELP). A bifunctional ELP-MerR fusion protein is created to enable the precipitation of plasmid DNA, designed to contain the MerR recognition sequence, by a simple temperature trigger. The protocol covers all stages of the process from the design of ELP-MerR fusion proteins and MerR-binding plasmids, to the isolation of plasmid DNA from Escherichia coli cultures after boiling lysis, the subsequent temperature-triggered precipitation of plasmid DNA-fusion protein complexes and final elution of plasmid DNA by mild heating. This protocol is well suited to laboratory research-scale applications, producing plasmid DNA of better purity and similar yield as one of the most commonly used laboratory methods, standard alkaline lysis (known as the midiprep procedure). The protocol takes approximately 30 min to obtain pure plasmid DNA from cell cultures using the temperature-triggered precipitation method.     

Replication protein A interactions with DNA. 1. Functions of the DNA-binding and zinc-finger domains of the 70-kDa subunit

Human replication protein A (RPA) is a multiple subunit single-stranded DNA-binding protein that is required for multiple processes in cellular DNA metabolism. This complex, composed of subunits of 70, 32, and 14 kDa, binds to single-stranded DNA (ssDNA) with high affinity and participates in multiple protein-protein interactions. The 70-kDa subunit of RPA is known to be composed of multiple domains: an N-terminal domain that participates in protein interactions, a central DNA-binding domain (composed of two copies of a ssDNA-binding motif), a putative (C-X2-C-X13-C-X2-C) zinc finger, and a C-terminal intersubunit interaction domain. A series of mutant forms of RPA were used to elucidate the roles of these domains in RPA function. The central DNA-binding domain was necessary and sufficient for interactions with ssDNA; however, adjacent sequences, including the zinc-finger domain and part of the N-terminal domain, were needed for optimal ssDNA-binding activity. The role of aromatic residues in RPA-DNA interactions was examined. Mutation of any one of the four aromatic residues shown to interact with ssDNA had minimal effects on RPA activity, indicating that individually these residues are not critical for RPA activity. Mutation of the zinc-finger domain altered the structure of the RPA complex, reduced ssDNA-binding activity, and eliminated activity in DNA replication.     

What can we learn from noncoding regions of similarity between genomes?

Background  

In addition to known protein-coding genes, large amounts of apparently non-coding sequence are conserved between the human and mouse genomes. It seems reasonable to assume that these conserved regions are more likely to contain functional elements than less-conserved portions of the genome.     

3,4-Dichloroaniline is detoxified and exported via different pathways in Arabidopsis and soybean

The metabolic fate of [UL-14C]-3,4-dichloroaniline (DCA) was investigated in Arabidopsis root cultures and soybean plants over a 48 h period following treatment via the root media. DCA was rapidly taken up by both species and metabolised, predominantly to N-malonyl-DCA in soybean and N-glucosyl-DCA in Arabidopsis. Synthesis occurred in the roots and the respective conjugates were largely exported into the culture medium, a smaller proportion being retained within the plant tissue. Once conjugated, the DCA metabolites in the medium were not then readily taken up by roots of either species. The difference in the routes of DCA detoxification in the two plants could be explained partly by the relative activities of the respective conjugating enzymes, soybean containing high DCA-N-malonyltransferase activity, while in Arabidopsis DCA-N-glucosyltransferase activity predominated. A pre-treatment of plants with DCA increased DCA-N-malonyltransferase activity in soybean but not in Arabidopsis, indicating differential regulation of this enzyme in the two plant species. This study demonstrates that DCA can undergo two distinct detoxification mechanisms which both lead to the export of conjugated metabolites from roots into the surrounding medium in contrast to the vacuolar deposition more commonly associated with the metabolism of xenobiotics in plants.     

Sticky anaphase aberrations after G2-phase arrest of gamma-irradiated human skin fibroblasts: TP53 independence of formation and TP53 dependence of consequences

We have studied the impact of TP53 status on the extent and nature of chromosome damage seen in human skin fibroblasts after gamma irradiation beyond the G1-phase checkpoint but prior to the G2-phase checkpoint. Mitotic cells were examined in the absence and presence of treatment with nocodazole and the yield of aberrations was scored as a function of time postirradiation. The results revealed substantially greater damage in the absence of nocodazole, indicating that damage was being masked in its presence. While metaphase aberrations were seen exclusively in the presence of nocodazole, anaphase aberrations were seen principally in its absence. Furthermore, these were mostly of an unseparated, or "sticky", type that showed separation of the chromatids in the centromeric region, indicating normal degradation of cohesin, with retention of adhesion further out on the chromatid arms. Using postirradiation BrdU labeling and the absence of nocodazole, we were able to identify mitotic figures up to the third postirradiation mitosis. Analysis of the data revealed that in cells wild-type for TP53 the aberrant anaphases were lost after the first postirradiation mitosis, although they were still found in gradually decreasing amounts into the second and third postirradiation mitoses in E6-expressing cells. The data indicate that the formation of these sticky anaphases is independent of TP53 status, an observation that is consistent with the TP53 independence of transient G2-phase arrest. However, the consequences of the formation of these lesions appear to be very different. In the case of cells wild-type for TP53 this is chronic G1-phase arrest, while in E6 cells it is anaphase catastrophe.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.     

Pénurie de technétium99m : des pistes pratiques pour gérer les périodes de crise dans les services de médecine nucléaire

Technetium^99m (^99mTc) shortage crisis regularly affect nuclear medicine activity and oblige the community to find solutions in order to perform most of the prescribed exams and avoid systematic substitutions by other non-nuclear medicine techniques. Firstly, some practical solutions can be set up in radiopharmacy departments such as using more than two generators together, realizing fractionated elutions, preparing radiopharmaceuticals with elutions providing from different generators… Then, it could be interesting to have a reflexion in nuclear medicine departments to convene patients the days when ^99mTc supply is sufficient, to pool some exams or to make substitutions with more available isotopes.