Exosporium components from endospores of Bacillus cereus ATCC 10876 were purified and separated by gel electrophoresis. Several of the proteins for which N‐terminal sequences were recovered were found to have homologues in protein databases which have been demonstrated to have enzymic activity in other organisms. Amongst these is a zinc metalloprotease, immune inhibitor A, already described in B. thuringiensis. This has been shown to be present in an active 73 kDa form on the exosporium of B. cereus. Other proteins associated with the exosporium include the molecular chaperone GroEL and a homologue of RocA (1‐pyrroline‐5‐carboxylate dehydrogenase (EC 1.5.1.12)) of B. subtilis. Although these are unlikely to represent integral structural proteins of the exosporium, the observation that they are selectively present in the spore surface layer suggests that this layer may have functional significance.

Galectins are members of a genetically related family of ␤-galactoside-binding lectins. At least eight distinct mammalian galectins have been identified. More distantly related, but still conserving amino acid residues critical for carbohydrate-binding, are galectins in chicken, eel, frog, nematode, and sponge. Here we report that galectins are also expressed in a species of fungus, the inky cap mushroom, Coprinus cinereus. Two dimeric galectins are expressed during fruiting body formation which are 83% identical to each other in amino acid sequence and conserve all key residues shared by members of the galectin family. Unlike most galectins, these have no N-terminal post-translational modification and no cysteine residues. We expressed one of these as a recombinant protein and studied its carbohydrate-binding specificity using a novel nonradioactive assay. Binding specificity has been well studied for a number of other galectins, and like many of these, the recombinant C. cinereus galectin shows particular affinity for blood group A structures. These results demonstrate not only that the galectin gene family is evolutionarily much older than previously realized but also that fine specificity for complex saccharide structures has been conserved. Such conservation implies that galectins evolved to perform very basic cellular functions, presumably by interaction with glycoconjugates bearing complex lactoside carbohydrates resembling blood group A.Galectins are animal lectins that are related in amino acid sequence and specifically bind to ␤-galactoside carbohydrates such as lactose (1). Members of this gene family all include a conserved carbohydrate-binding domain but vary in inclusion of other domains and in tissue expression patterns. More distantly related, but conserving critical amino acid residues involved in carbohydrate-binding, are galectins in chicken, eel, frog, nematode, and sponge (2).Although galectins have been studied for 20 years now, physiological functions for these proteins have not yet been clearly established. Their affinity for oligosaccharides found on glycoconjugates on cell surfaces or in extracellular matrix has suggested that galectins function extracellularly by binding to such ligands. Indeed, certain galectins have particular affinity for specific glycoprotein ligands, such as polylactosamine chains on laminin (1, 3). When added to cells or overexpressed after transfection, galectins can have major effects on cell adhesion, proliferation, apoptosis, metastasis, and immune function (1-6). However, evidence has also been presented for intracellular functions of galectins, for instance in message splicing (7) or as nuclear proteins (8). Therefore, efforts are being directed at exploring the evolutionary origin of galectins in the hope that their functions will be easier to define in simple model organisms.Here we report that a species of fungus, the inky cap mushroom, Coprinus cinereus, expresses two lectins related in sequence and carbohydrate-binding specificity to other galectins. T...

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.

An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.