Use of a restriction enzyme-digested PCR product as substrate for helicase assays

DNA helicases play essential roles in many cellular processes. The currently available techniques to generate substrates for helicase assays are fairly complicated and need some expertise not available in all laboratories. Here, a PCR-based method to generate a substrate for a helicase assay is described, and its application for several archaeal, bacterial and viral enzymes is demonstrated

An unusual presentation of postaxial polydactyly of the foot

[Abstract] A 6-month-old Caucasian baby is described with a postaxial polydactyly of the letf foot. Radiographic examination revealed the accessory digit was composed of soft tissue, some with a tiny osseous element, originated from around the metatarsophalangeal joint, defined by floating type (FT). The parents had consistent difficulty putting shoes. We encountered an exceedingly rare presentation of FT, to our inspection, had neither been previously related in published studies. To the best of our knowledge, this represents the unusual case of congenital deformity lesion on the left foot to be reported in the medical literature

A new screening method for selection of desired recombinant plasmids in molecular cloning

One of the problems in cloning process is the low concentration of gene fragment and vector following gel extraction stage which may lead to decreased likelihood of effective ligation. Regarding the facts of this study, after digestion process, the products directly were subjected to ligation. Due to the presence of three antibiotic resistance genes in the reaction, a new strategy based on design of selective media containing different antibiotics was used for selecting the desired colonies. The results of this study indicated that skipping gel extraction process could result in a successful, simple and quick cloning process with high efficiency.Keywords: Cloning, gel extraction, vector, screening, antibiotic resistance gene

WebGeSTer DB-a transcription terminator database

We present WebGeSTer DB, the largest database of intrinsic transcription terminators (http://pallab .serc.iisc.ernet.in/gester). The database comprises of a million terminators identified in 1060 bacterial genome sequences and 798 plasmids. Users can obtain both graphic and tabular results on putative terminators based on default or user-defined parameters. The results are arranged in different tiers to facilitate retrieval, as per the specific requirements. An interactive map has been incorporated to visualize the distribution of terminators across the whole genome. Analysis of the results, both at the whole-genome level and with respect to terminators downstream of specific genes, offers insight into the prevalence of canonical and non-canonical terminators across different phyla. The data in the database reinforce the paradigm that intrinsic termination is a conserved and efficient regulatory mechanism in bacteria. Our database is freely accessible

Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins

International audienceThe generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals

Time Variations of Macrostickies and Extractable Stickies Concentrations in Deinking

The stickies content, both macrostickies and stickies extractable in a solvent, was determined for samples taken at short time intervals from deinking lines, producing deinked pulp for newsprint production. The study was carried out at three mills on different continents, with each having a different source of recycled paper as raw material. The short-term variations in extractable stickies in the incoming raw material were quite extreme, with differences of 100% being seen within hours. Despite this, the final deinked pulp contained fewer sudden variations and had no correlation to the incoming stickies content. While the raw material appeared to affect the incoming stickies content, a well-optimized deinking line was able to buffer the raw material variability, and the final stickies content was more dependent on the deinking process. This result was seen for the two mills examined for this phenomenon, despite a different raw material supply. Macrostickies were found to exhibit the same tendencies, although with smaller and less sudden variations. However, the variations of macrostickies and extractable stickies never correlated, even when both were measured for the same pulp fraction, thus confirming that solvent extraction is not an appropriate method for the determination of macrostickies and is more a reflection of microstickies

L-arginine recognition by yeast arginyl-tRNA synthetase.

The crystal structure of arginyl-tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl-tRNA synthetase (aaRS), with L-arginine bound to the active site has been solved at 2.75 A resolution and refined to a crystallographic R-factor of 19.7%. ArgRS is composed predominantly of alpha-helices and can be divided into five domains, including the class I-specific active site. The N-terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition. The C-terminal domain, which is the putative anticodon-binding module, displays an all-alpha-helix fold highly similar to that of Escherichia coli methionyl-tRNA synthetase. While ArgRS requires tRNAArg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L-arginine binding. All H-bond-forming capability of L-arginine is used by the protein for the specific recognition. The guanidinium group forms two salt bridge interactions with two acidic residues, and one H-bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences. This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles. The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs

Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain

DNA helicases catalyze separation of double-helical DNA into its complementary single strands, a process essential for DNA replication, recombination, and repair. The Escherichia coli Rep protein, a superfamily 1 DNA helicase, functions in DNA replication restart and is required for replication of several bacteriophages. Monomers of Rep do not display helicase activity in vitro; in fact, DNA unwinding requires Rep dimerization. Here we show that removal of the 2B subdomain of Rep to form RepΔ2B activates monomer helicase activity, albeit with limited processivity. Although both full length Rep and RepΔ2B monomers can translocate with 3′ to 5′ directionality along single-stranded DNA, the 2B subdomain inhibits the helicase activity of full length Rep. This suggests an autoregulatory mechanism for Rep helicase, which may apply to other nonhexameric helicases, whereby helicase activity is regulated by the rotational conformational state of the 2B subdomain; formation of a Rep dimer may relieve autoinhibition by altering the 2B subdomain orientation