An ultra-rapid cryo-technique for complex organisms

The soil nematode Caenorhabditis elegans is an excellent research model in cell biology, human disease and developmental studies. In this study, a novel cryopreservation technique based on a rapid cooling procedure, previously established for juveniles, was applied to adult-worms. Here we demonstrated that adults of C. elegans, a complex metazoan organism, survive to a rapid cooling and storage in liquid nitrogen (-196 °C) with a very high survival percentage (85%). The procedure relies on a Low CryoProtectant Technique (LCPT) and Ultra Rapid Cooling (URC). The high cooling rate is achieved through the reduction of sample volumes and the effectiveness of a nylon carrier. Our technique complies with the requirements for vitrification to occur. The main distinctive characters of this cryopreservation technique compared to other methods, e.g. Slow Freezing and Vitrification, are presented. Our results show that this cryopreservation method is valid for both unicellular and multicellular organisms; it is suitable for short or long time storage in liquid-nitrogen. This technique promises to be a unique and simpler method for cryostorage of cells, organisms and tissues

Restriction enzyme and DNA hybridization analysis of cellulolytic Streptomyces isolates of different origin.

Streptomyces rochei A2 endoglucanase (eglS) and β-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities. The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S. rochei A2. The DNA from all strains, except one, hybridized with the bgs1 probe and one strain showed the same restriction pattern as seen in S. rochei A2. The sequence localized by the eglS probe in S. thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E. coli in plasmids pTAE and pCSF203, respectively. The restriction maps showed that the cloned genes were identical to eglS and bgs1. The restriction enzyme analysis of genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments

Bestimmung der intraspezifischen Variabilität von Isolaten des Kiefernholznematoden Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) mittels ISSR- und RAPD-Fingerprints

The pinewood nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease. In order to trace the origin of its recently introduced Portuguese population, two PCR-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR), were used to determine genetic relationships among 30 B. xylophilus isolates from the Usa, Canada, Japan, China, South Korea and Portugal. Fingerprints obtained with both methods detected a reduced genetic variation of introduced popula- tions as compared to native North American populations. Cluster analyses of genetic distances between isolates were carried out and bootstrap dendrograms were constructed. The results indicated that founders of the Portuguese popula- tions most likely were translocated one or two times to Portugal from their colonized sites in East Asia, but not from their native habitats in North America