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GDP-L-fucose is the activated nucleotide sugar form of L-fucose, which is a constituent of many structural polysaccharides and glycoproteins in various organisms. The de novo synthesis of GDP-L-fucose from GDP-D-mannose encompasses three catalytic steps, a 4,6-dehydration, a 3,5-epimerization, and a 4-reduction. The mur1 mutant of Arabidopsis is deficient in L-fucose in the shoot and is rescued by growth in the presence of exogenously supplied L-fucose. Biochemical assays of the de novo pathway for the synthesis of GDP-L-fucose indicated that mur1 was blocked in the first nucleotide sugar interconversion step, a GDP-Dmannose-4,6-dehydratase. An expressed sequence tag was identified that showed significant sequence similarity to proposed bacterial GDP-D-mannose-4,6-dehydratases and was tightly linked to the mur1 locus. A full-length clone was isolated from a cDNA library, and its coding region was expressed in Escherichia coli. The recombinant protein exhibited GDP-D-mannose-4,6-dehydratase activity in vitro and was able to complement mur1 extracts in vitro to complete the pathway for the synthesis of GDP-L-fucose. All seven mur1 alleles investigated showed single point mutations in the coding region for the 4,6-dehydratase, confirming that it represents the MUR1 gene.L-Fucose (6-deoxy-L-galactose) is a monosaccharide found in a diverse array of organisms. The sugar is a known component of bacterial lipopolysaccharides, mammalian and plant glycoproteins, and polysaccharides of plant cell walls such as xyloglucan and rhamnogalacturonans I and II. The precise function of L-fucose within these polysaccharides is not clear, but it may stabilize conformations of xyloglucan, which can efficiently bind to cellulose microfibrils (1), possibly aiding in cell wall integrity. Furthermore, xyloglucan fucosylation is essential for the biological activity of some xyloglucan-derived oligosaccharides (2). The pathway for the synthesis of L-fucose has been studied biochemically, but genes for the corresponding enzymes have not been cloned from any eukaryote.GDP-L-fucose (guanosine-diphospho-L-fucose) is the activated form of this sugar, synthesized de novo from GDP-Dmannose via a three-step mechanism or through a salvage pathway involving phosphorylation of free L-fucose and subsequent nucleoside-diphosphate attachment (3-5). The de novo pathway for GDP-L-fucose production is shown in Fig. 1. The first step is catalyzed by GDP-D-mannose-4,6-dehydratase and involves the formation of the intermediate GDP-4-keto-6-deoxy-D-mannose, which is then used in the second and third steps of the pathway by 3,5-epimerase and 4-reductase activities to yield GDP-L-fucose. The pathway was initially elucidated in bacteria but has since been characterized in mammalian and plant systems (6)(7)(8)(9)(10)(11).Recently an L-fucose-deficient cell wall mutant, mur1, was isolated from Arabidopsis thaliana and characterized phenotypically (12). Eight recessive mur1 alleles were obtained from this screen, most of which exhibit 50-to 200-fold reducti...

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With the increasing number of eukaryotic genomes available, high-throughput automated tools for identification of regulatory DNA sequences are becoming increasingly feasible. Several computational approaches for the prediction of regulatory elements were recently developed. Here we combine the prediction of clusters of binding sites for transcription factors with context information taken from genome annotations. Target Explorer automates the entire process from the creation of a customized library of binding sites for known transcription factors through the prediction and annotation of putative target genes that are potentially regulated by these factors. It was specifically designed for the well-annotated Drosophila melanogaster genome, but most options can be used for sequences from other genomes as well. Target Explorer is available at http://trantor.bioc.columbia.edu/Target_Explorer/

SummaryBecause of the limited lysine content in corn grain, synthetic lysine supplements are added to corn meal-based rations for animal feed. The development of biotechnology, combined with the understanding of plant lysine metabolism, provides an alternative solution for increasing corn lysine content through genetic engineering. Here, we report that by suppressing lysine catabolism, transgenic maize kernels accumulated a significant amount of lysine. This was achieved by RNA interference (RNAi) through the endosperm-specific expression of an inverted-repeat (IR) sequence targeting the maize bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharopine dehydrogenase (ZLKR/SDH). Although plant-short interfering RNA (siRNA) were reported to lack tissue specificity due to systemic spreading, we confirmed that the suppression of ZLKR/SDH in developing transgenic kernels was restricted to endosperm tissue. Furthermore, results from our cloning and sequencing of siRNA suggested the absence of transitive RNAi.These results support the practical use of RNAi for plant genetic engineering to specifically target gene suppression in desired tissues without eliciting systemic spreading and the transitive nature of plant RNAi silencing.