Quantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit

The eukaryotic cytosolic chaperonin CCT is an essential ATP-dependent protein folding machine whose action is required for folding the cytoskeletal proteins actin and tubulin, and a small number of other substrates, including members of the WD40-propellor repeat-containing protein family. An efficient purification protocol for CCT from Saccharomyces cerevisiae has been developed. It uses the calmodulin binding peptide as an affinity tag in an internal loop in the apical domain of the CCT3 subunit, which is predicted to be located on the outside of the double-ring assembly. This purified yeast CCT was used for a novel quantitative actin-folding assay with human beta-actin or yeast ACT1p protein folding intermediates, Ac(I), pre-synthesised in an Escherichia coli translation system. The formation of native actin follows approximately a first-order reaction with a rate constant of about 0.03 min(-1). Yeast CCT catalyses the folding of yeast ACT1p and human beta-actin with nearly identical rate constants and yields. The results from this controlled CCT-actin folding assay are consistent with a model where CCT and Ac(I) are in a binding pre-equilibrium with a rate-limiting binding step, followed by a faster ATP-driven processing to native actin. In this pure in vitro system, the human beta-actin mutants, D244S and G150P, show impaired folding behaviour in the manner predicted by our sequence-specific recognition model for CCT-actin interaction.     

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.