Size variation of rDNA clusters in the yeasts Saccharomyces cerevisiea and Schizosaccharomyces pombe

Summary The higher-order organization of rRNA genes was investigated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We used pulsed-field gel electrophoresis (PFGE) in combination with frequent cutter endonucleases having no recognition sites within rDNA repeating units to characterize tandem arrays of ribosomal genes in these two species. Large variations in rDNA cluster length were detected in various S. cerevisiae and S. pombe strains commonly used as PFGE molecular weight markers. This wide range of variability implies that the sizes currently assessed for chromosomes bearing rRNA genes in these organisms are unreliable since they may vary within strains by several hundreds of kilobase pairs, depending on the size of the tandem arrays of rRNA genes. Consequently, there is now a lack of reliable PFGE size standards between 1.6 Mb and 4.5 Mb, even when established yeast strains with calibrated chromosomes are used.     

Bacterial diversity in the bulk soil and rhizosphere fractions of Lolium perenne and Trifolium repens as revealed by PCR restriction analysis of 16S rDNA

The rhizosphere of Trifolium repens and Lolium perenne was divided into three fractions: the bulk soil, the soil adhering to the roots and the washed roots (rhizoplane and endorhizosphere). After isolation and purification of DNA from these fractions, 16S rDNA was amplified by PCR and cloned to obtain a collection of 16S rRNA genes representative of the bacterial communities of these three fractions. The genes were then characterized by PCR restriction analysis. Each different profile was used to define an operational taxonomic unit (OTU). The numbers of OTUs and the numbers of clones among these OTUs allowed to calculate a diversity index. The number of OTUs decreased as root proximity increased and a few OTUs became dominant, resulting in a lower diversity index. In the root fraction of T. repens, the restriction profile of the dominant OTU matched the theoretical profile of the 16S rRNA gene of Rhizobium leguminosarum. This study showed that plant roots create a selective environment for microbial populations.     

Calretinin and calretinin-22k increase resistance toward sodium butyrate-induced differentiation in CaCo-2 colon adenocarcinoma cells.

Calretinin (CR) and the alternatively spliced form calretinin-22k (CR-22k) are members of the EF-hand family of Ca(2+)-binding proteins (CaBPs). CR is expressed in more than 60% of poorly differentiated human colon tumors and both isoforms are present in several colon carcinoma cell lines (e.g., WiDr). They are absent in normal enterocytes and in well-differentiated adenocarcinoma cell lines such as CaCo-2. Calretinins are thought to act as Ca(2+) buffers and to be involved in the regulation of Ca(2+)-dependent processes. Down-regulation of calretinins in WiDr cells by antisense oligonucleotides leads to growth inhibition and treatment with sodium butyrate (NaBt, an inducer of differentiation) leads to a blockage of the cell cycle and, in parallel, to down-regulation of CR. It has been proposed that CR and/or CR-22k may be involved in maintaining the undifferentiated phenotype of WiDr cells and contributing to the transformation of enterocytes. Expression levels and distribution of CR-22k were investigated in WiDr cells. CR-22k was down-regulated in NaBt-treated cells and the normally cytoplasmic protein was preferentially localized in the nucleus either as a result of translocation or selective nuclear maintenance, a process more pronounced than in the case of CR. To compare functional differences of calretinins, CR-negative Caco-2 cells were stably transfected with cDNAs encoding CR or CR-22k. Cell growth of CR-transfected cells was increased, an effect that was not observed in CR-22k-transfected ones. The CR-expressing clones were almost completely resistant to treatment with 0.5 mM NaBt, a concentration significantly reducing cell growth in control cells. The same effect was obtained in the CR-22k-expressing clones, although to a lesser extent. This implicates that expression of CR and/or CR-22k in colon tumor cells may contribute to tumorigenesis by blocking differentiation-promoting signals.     

Influence of an Elevated Atmospheric CO2 Content on Soil and Rhizosphere Bacterial Communities Beneath Lolium perenne and Trifolium repens under Field Conditions

Abstract The increase in atmospheric CO₂ content alters C₃ plant photosynthetic rate, leading to changes in rhizodeposition and other root activities. This may influence the activity, the biomass, and the structure of soil and rhizosphere microbial communities and therefore the nutrient cycling rates and the plant growth. The present paper focuses on bacterial numbers and on community structure. The rhizospheres of two grassland plants, Lolium perenne (ryegrass) and Trifolium repens (white clover), were divided into three fractions: the bulk soil, the rhizospheric soil, and the rhizoplane–endorhizosphere. The elevated atmospheric CO₂ content increased the most probable numbers of heterotrophic bacteria in the rhizosphere of L. perenne. However, this effect lasted only at the beginning of the vegetation period for T. repens. Community structure was assessed after isolation of DNA, PCR amplification, and construction of cloned 16S rDNA libraries. Amplified ribosomal DNA restriction analysis (ARDRA) and colony hybridization with an oligonucleotide probe designed to detect Pseudomonas spp. showed under elevated atmospheric CO₂ content an increased dominance of pseudomonads in the rhizosphere of L. perenne and a decreased dominance in the rhizosphere of T. repens. This work provides evidence for a CO₂-induced alteration in the structure of the rhizosphere bacterial populations, suggesting a possible alteration of the plant-growth-promoting-rhizobacterial (PGPR) effect. Received: 14 December 1998; Accepted: 31 March 1999