Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulinregulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.Glucose transport into mammalian muscle and fat cells is an important step in insulin action and is critical for the maintenance of glucose homeostasis within the body (1). In mammalian muscle and fat cells, insulin stimulation activates a phosphorylation cascade, which in turn causes intracellular vesicles that contain the glucose transporter GLUT4, 4 to translocate to the plasma membrane (PM) and fuse (2, 3). In the basal state GLUT4 is distributed between the endosomal system, the trans-Golgi network (TGN), and a GLUT4 storage vesicle (GSV) compartment that is highly insulin-responsive (4 -6).The protein kinase Akt is activated in response to insulin and plays a critical role in GLUT4 translocation (1, 7). However, the link between the insulin signaling pathway and GLUT4 translocation is not fully understood. The insulin-dependent movement of GLUT4 vesicles to the PM is an Akt-independent process, and this is followed by an Aktdependent step likely involving the docking and fusion of vesicles with the PM (7-9). The mechanism by which Akt controls the docking and fusion of GLUT4 vesicles with the PM is not known. However, it was previously shown that a Rab GTPase-activating protein (RabGAP) known as AS160 is phosphorylated by Akt in response to insulin (10). How AS160 functions in GLUT4 trafficking and its cognate Rab proteins are not known. The role of a variety of Rab proteins in GLUT4 trafficking including Rab3d, Rab4, Rab5, and Rab11 has been examined (11-16). However, although these Rab proteins may participate in some aspects of GLUT4 trafficking, no compelling evidence for specific involvement in the insulin-regulated trafficking of GLUT4 has been found.In this study we describe four key findings that add to our understanding of GLUT4 traffic...

Many strains of E. coli K12 restrict DNA containing cytosine methylation such as that present in plant and animal genomes. Such restriction can severely inhibit the efficiency of cloning genomic DNAs. We have quantitatively evaluated a total of 39 E. coli strains for their tolerance to cytosine methylation in phage and plasmid cloning systems. Quantitative estimations of relative tolerance to methylation for these strains are presented, together with the evaluation of the most promising strains in practical recombinant cloning situations. Host strains are recommended for different recombinant cloning requirements. These data also provide a rational basis for future construction of 'ideal' hosts combining optimal methylation tolerance with additional advantageous mutations.

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Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.