Extracellular DNA and Type IV pili mediate surface attachment by Acidovorax temperans.

Extracellular DNA can play a structural role in the microbial environment. Here evidence is presented that an environmental isolate of Acidovorax temperans utilises extracellular DNA for intercellular and cell-surface attachment and that Type IV pili and electrostatic interactions play a role in this interaction. Preliminary attempts to isolate and purify extracellular polysaccharides from A. temperans strain CB2 yielded significant amounts of DNA raising the question of whether this molecule was present as a structural component in the extracellular matrix. The role of DNA in attachment was indicated by experiments in which the addition of DNase to liquid medium inhibited the attachment of Acidovorax to glass wool. A Tn5 insertional mutant, lacking Type IV pili, was unable to initiate attachment. Addition of DNase caused rapid detachment of bound cells, but no detachment occurred when proteinase, RNase or inactivated DNase were used. Addition of MgCl(2) also caused significant detachment, supporting the possible mechanistic role of electrostatic interactions in the attachment process. Although attachment was apparent in early to mid-log phase growth, surprisingly DNA was not detected in the culture supernatant until late stationary phase and coincided with an appreciable loss of cell viability. This suggests that during log-phase growth attachment is mediated by eDNA that is released in low quantities and/or is highly localised within the extracellular matrix and also that stationary phase DNA release through widespread cell lysis may be a separate and unrelated event.

Global gene expression analysis of apple fruit development from the floral bud to ripe fruit.

BACKGROUND: Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45-55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple. RESULTS: Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes. CONCLUSION: Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development.

Technology for high-throughput screens: the present and future using zebrafish.

The zebrafish is a popular vertebrate model organism with similar organ systems and gene sequences to humans. Zebrafish embryos are optically transparent enabling organ visualisation, which can be complemented with gene expression analysis at the transcript and protein levels. Furthermore, zebrafish can be treated with small molecules and drugs in a microtitre plate format for high-throughput analysis and for the identification and validation of drugs. High-throughput methodologies for use in zebrafish include phenotype-based visualisation, transcript studies using low-density DNA microarrays and proteomic analysis. These technologies offer significant whole-organism biological value in the drug discovery and drug development pipeline.

Global gene expression analysis in the zebrafish: the challenge and the promise.

Microarray technology allows the simultaneous changes in multiple gene expression to be determined as part of a drug discovery platform, or to understand the biological mechanisms of disease. In terms of both of these objectives, those studying the zebrafish as a complement to mouse-based research are only now able to entertain the construction of microarrays due to recent progress in sequencing the zebrafish genome. The advantage of the late entry of zebrafish to the microarray field offers the means of immediately adopting best practice, while also appreciating that what is currently on offer can be improved.:

Chemical discovery and global gene expression analysis in zebrafish.

The zebrafish (Danio rerio) provides an excellent model for studying vertebrate development and human disease because of its ex utero, optically transparent embryogenesis and amenability to in vivo manipulation. The rapid embryonic developmental cycle, large clutch sizes and ease of maintenance at large numbers also add to the appeal of this species. Considerable genomic data has recently become publicly available that is aiding the construction of zebrafish microarrays, thus permitting global gene expression analysis. The zebrafish is also suitable for chemical genomics, in part as a result of the permeability of its embryos to small molecules and consequent avoidance of external confounding maternal effects. Finally, there is increasing characterization and analysis of zebrafish models of human disease. Thus, the zebrafish offers a high-quality, high-throughput bioassay tool for determining the biological effect of small molecules as well as for dissecting biological pathways.