Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

Effect of some biotic factors on microbially-induced calcite precipitation in cement mortar.

Sporosarcina pasteurii, a common soil bacterium has been tested for microbial treatment of cement mortar. The present study also seeks to investigate the effects of growth medium, bacterial concentration and different buffers concerning the preparation of bacterial suspensions on the compressive strength of cement mortar. Two growth media, six different suspensions and two bacterial concentrations were used in the study. The influence of growth medium on calcification efficiency of S. pasteurii was insignificant. Significant improvement in the compressive as well as the tensile strength of cement mortar was observed. Microbial mineral precipitation visualized by Scanning Electron Microscopy (SEM) shows fibrous material that increased the strength of cement mortar. Formation of thin strands of fillers observed through SEM micrographs improves the pore structure, impermeability and thus the compressive as well as the tensile strengths of the cement mortar. The type of substrate and its molarity have a significant influence on the strength of cement mortar.

Bending of DNA segments with Saccharomyces cerevisiae autonomously replicating sequence activity, isolated from basidiomycete mitochondrial linear plasmids.

Previous studies have indicated that DNA bending is a general structural feature of sequences (ARSs) from cellular DNAs of yeasts and nuclear and mitochondrial genomic DNAs of other eukaryotes that are capable of autonomous replication in Saccharomyces cerevisiae. Here we showed that bending activity is also tightly associated with S. cerevisiae ARS function of segments cloned from mitochondrial linear DNA plasmids of the basidiomycetes Pleurotus ostreatus and Lentinus edodes. Two plasmids, designated pLPO2-like (9.4 kb), and pLPO3 (6.6 kb) were isolated from a strain of P. ostreatus. A 1029 bp fragment with high-level ARS activity was cloned from pLPO3 and it contained one ARS consensus sequence (A/T)TTTAT(A/G)TTT(A/T) indispensable for activity and seven dispersed ARS consensus-like (10/11 match) sequences. A discrete bent DNA region was found to lie around 500 bp upstream from the ARS consensus sequence (T-rich strand). Removal of the bent DNA region impaired ARS function. DNA bending was also implicated in the ARS function associated with a 1430 bp fragment containing three consecutive ARS consensus sequences which had been cloned from the L. edodes plasmid pLLE1 (11.0 kb): the three consecutive ARSs responsible for high-level ARS function occurred in, and immediately adjacent to, a bent DNA region. A clear difference exists between the two plasmid-derived ARS fragments with respect to the distance between the bent DNA region and the ARS consensus sequence(s).