Differentiation of Acidithiobacillus ferrooxidans and A-thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli. (C) 2004 Elsevier SAS. All rights reserved.155755956

Cisplatin and siRNA interference with structure and function of Wnt-5a mRNA: design and in vitro evaluation of targeting AU-rich elements in the 3' UTR.

Wnt-5a is a secreted glycoprotein which has been shown to be involved in the regulation of cell adhesion and motility, processes which are of importance in metastasis formation by cancer cells. We here present an initial study aiming at evaluating whether small interfering RNA (siRNA) in combination with cisplatin can be used to modulate protein expression levels under in vitro conditions. For this purpose, an AU-rich region corresponding to the initial 260 bases of the Wnt-5a 3' untranslated region was chosen as the target. The effect of four different siRNAs was evaluated by analysis of protein suppression levels in rabbit reticulocyte lysate (RRL) and an immortalized noncancerous mammary epithelial (HB2) cell line by monitoring the activity of transiently expressed luciferase. The specificity and kinetics for hybridization of the siRNA with the messenger RNA target were followed by digestion techniques and analysis by polyacrylamide gel electrophoresis. Specific and temperature-dependent hybridization was observed, with a half-life of approximately 0.5 h at 4 degrees C. Significant downregulation of luciferase activity was obtained in the micromolar and nanomolar range, for RRL and HB2, respectively. In addition, the downregulation of protein production caused by addition of cisplatin could be further potentiated by addition of siRNA in a selective manner. The latter observation suggests that combined use of cisplatin and siRNA could be a method to decrease therapeutically used cisplatin concentrations. Thus, toxic side effects could be minimized while key proteins are targeted in a highly specific manner