SummaryThe opuE gene from Bacillus subtilis encodes a transport system (OpuE) for osmoprotective proline uptake and is expressed from two osmoregulated promoters: opuE P-1 recognized by the vegetative sigma factor A ( A ) and opuE P-2 dependent on the stress-induced transcription factor sigma B ( B ). The contributions of these two promoters to osmoregulation of opuE were analysed. Genetic studies using chromosomal opuE-treA operon fusions revealed that opuE transcription is rapidly induced after an osmotic upshock. proportion to the external osmolarity and was maintained at high levels. Moreover, both promoters exhibited a different response to the osmoprotectant glycine betaine in the medium. Our results suggest that at least two different signal transduction pathways operate in B. subtilis to communicate osmotic changes in the environment to the transcription apparatus of the cell.

The agricultural biotechnology industry applies polymerase chain reaction (PCR) technology at numerous points in product development. Commodity and food companies as well as third-party diagnostic testing companies also rely on PCR technology for a number of purposes. The primary use of the technology is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of PCR analysis and its application to the testing of grains. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effect they may have on the accuracy of the PCR analytical results.

Various countries have established regulations that stipulate the labeling of agricultural commodities, feed, and food products that contain or are made from genetically modified (GM) material or that contain adventitious GM material in amounts that exceed certain threshold levels. While regulations in some countries refer to GM material on a weight per weight (w/w) percentage, the currently applied detection methods do not directly measure the w/w percentage of the GM material. Depending on the particular method and the sample matrix it is applied to, the conversion of analytical results to a w/w percentage is challenging or not possible. The first rapid PCR system for GM maize detection on a single kernel basis has been developed. The equipment for the grinding of individual kernels and a silica membrane-based 96-well DNA extraction kit were both significantly revised and optimized for this particular purpose, respectively. We developed a multiplex real-time PCR method for the rapid quantification of GM DNA sequences in the obtained DNA solutions. In addition, a multiplex qualitative PCR detection method allows for the simultaneous detection of different GM maize traits in each kernel and thereby for identification of individual kernels that contain a combination of two or more GM traits. Especially for grain samples that potentially contain combined-trait GM maize kernels, the proposed methods can deliver informative results in a rapid, precise, and reliable manner.

"Genetically modified" (GM) or "biotech" crops have been the most rapidly adopted agricultural technology in recent years. The development of a GM crop encompasses trait identification, gene isolation, plant cell transformation, plant regeneration, efficacy evaluation, commercial event identification, safety evaluation, and finally commercial authorization. This is a lengthy, complex, and resource-intensive process. Crops produced through biotechnology are the most highly studied food or food component consumed. Before commercialization, these products are shown to be as safe as conventional crops with respect to feed, food, and the environment. This paper describes this global process and the various analytical tests that must accompany the product during the course of development, throughout its market life, and beyond.

Because of the increasing use of maize hybrids with genetically modified (GM) stacked events, the established and commonly used bulk sample methods for PCR quantification of GM maize in non-GM maize are prone to overestimate the GM organism (GMO) content, compared to the actual weight/weight percentage of GM maize in the grain sample. As an alternative method, we designed and assessed a group testing strategy in which the GMO content is statistically evaluated based on qualitative analyses of multiple small pools, consisting of 20 maize kernels each. This approach enables the GMO content evaluation on a weight/weight basis, irrespective of the presence of stacked-event kernels. To enhance the method's user-friendliness in routine application, we devised an easy-to-use PCR-based qualitative analytical method comprising a sample preparation step in which 20 maize kernels are ground in a lysis buffer and a subsequent PCR assay in which the lysate is directly used as a DNA template. This method was validated in a multilaboratory collaborative trial.

The POU proteins Brn-3a and Brn-3b belong to a family of DNA binding transcription factors that share stretches of extensive homology. Both Brn-3a and Brn-3b are expressed as shorter and longer isoforms. The long form of Brn-3a is able to oncogenically transform primary fibroblasts. By contrast, the short form of Brn-3b (Brn-3b(s)) cannot transform fibroblasts but is able to specifically inhibit the transforming activity of Brn-3a(l). Moreover, Brn-3a(l) can act as a transcriptional transactivator, while Brn-3b(s) is not only unable to do so but in addition specifically inhibits the transactivating activity of Brn-3a(l). Here, we show that the opposite and antagonistic activities of Brn-3a(l) and Brn-3b(s) proteins are due to their different DNA binding properties; Brn-3a(l) but not Brn-3b(s) can form stable complexes with several octamer-related target DNA sequences. The presence of Brn-3b(s) completely inhibits the binding of Brn-3a(l) to DNA by preventing the formation of Brn-3a(l)-DNA complexes as well as by disrupting preformed complexes. Experiments with GST fusion proteins and in vitro binding studies suggest that the inhibition of Brn-3a(l) activity by Brn-3b(s) occurs via direct interaction of the two transcription factors in solution. Therefore, we hypothesize that Brn-3b(s) can act as a direct antagonist of Brn-3a(l) by inhibiting its DNA binding through the formation of an inactive hetero-oligomeric complex.The POU domain represents a structural entity that was first recognized as a conserved motif in the transcription factors Pit-1, Oct-1, Oct-2, and Unc-86. All four proteins act as transcriptional regulators and play an important role in tissuespecific gene regulation, assuring the proper development and differentiation of target cells (for reviews, see Refs. 1 and 2). The POU domain is subdivided into a POU specific domain and a POU homeodomain; both subdomains are separated by a short linker sequence and are responsible for the DNA binding activity of POU transcription factors. In contrast to classical homeodomain transcription factors, POU proteins seem to recognize longer DNA motifs, which represent variants of the octamer consensus sequence ATGCAAAT (3-6).The POU family of transcription factors has grown substantially after a successful search for related genes based on PCR 1 experiments with degenerated oligonucleotides (7) and has been subdivided into six classes named POU I-VI (1, 2). Among others, the rat homologue of the Caenorhabditis elegans gene encoding Unc-86 was isolated and termed brn-3a (7) due to its predominant expression in rat brain. Independently, a human homologue of unc-86, then called RDC-1, was isolated from a neuroepithelioma cDNA library and found to be identical to brn-3a (8). The analysis of the newly isolated brn-3a gene revealed also the existence of two other related genes, brn-3b and brn-3c (9 -16). brn-3a, brn-3b, and brn-3c have also been termed brn-3.0, brn-3.2, and brn-3.1, respectively (2). All three brn-3 genes are differentially expressed during the develop...

To monitor pineal function in the Djungarian hamster (Phodopus sungorus), we measured the urinary excretion of the melatonin metabolite 6-sulphatoxymelatonin (aMT6s) at 3-hr intervals by radioimmunoassay. Hamsters maintained in either long photoperiod (LP, LD 16:8) or short photoperiod (SP, LD 8:16) showed marked daily rhythms in aMT6s excretion, with elevated levels during the dark phase. In both photoperiods, we found large interindividual differences, mainly in the amplitude of the signal. However, the amplitude as well as the duration of nocturnal aMT6s excretion was higher in SP than in LP. Light exposure at night (180 mW/m2, 30 min) caused a decrease in aMT6s excretion, indicating that the pineal gland is the major source of urinary aMT6s. Moreover, there was a significant correlation between nocturnal pineal/plasma melatonin contents and 24-hr aMT6s excretion. We conclude that, measurements of aMT6s provide a valid and quantitative index of pineal melatonin synthesis in this hamster species. As an advantage in determining pineal melatonin contents, this approach will allow noninvasive long-term studies of individual animals under varying environmental conditions.

In 2005 it was reported that the genetically modified (GM) maize strain or "event" called Bt10 had been distributed inadvertently in the United States over the previous 4 years. In order to ensure that grain for food and feed production did not contain trace amounts of Bt10 maize and complied with the applicable regulation, highly sensitive and specific detection of Bt10 maize was required. Accordingly, we developed a novel qualitative PCR system for specific detection of Bt10 maize. Moreover, we amply evaluated the performance characteristics of two PCR systems, our own and the one provided by the developer of Bt10, Syngenta Co. Ltd. It was confirmed that both of the qualitative PCR systems can specifically detect Bt10 maize, and the results of a single-laboratory examination suggested that the limit of detection was approximately less than 0.05% for both methods. To evaluate the reproducibility of the methods, we organized an interlaboratory study with the participation of 6 laboratories and analysis of 240 blind test samples. In this paper, we report, for the first time, the statistical analysis of the qualitative PCR data obtained from the interlaboratory study. The results of this analysis also revealed that there was no significant difference in the sensitivity between the two aforementioned methods and that the limit of detection of both the methods was less than 0.05%. Thus, we conclude that both of the methods are equally suitable for correct identification and sensitive detection of the unapproved GM maize Bt10 event in test samples.