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Regeneration in metazoans is one of the least understood biological processes. The lack of progress towards understanding this phenomenon at the molecular level is due in part to the poor regenerative abilities of the organisms traditionally used for developmental studies. Among the metazoans, planarians are known to have remarkable regenerative properties. In addition, flatworms are widely held to be among the simplest organisms possessing three tissue layers (triploblasts), bilateral symmetry, cephalization and complex organ systems. The combination of their regenerative properties and morphological simplicity make planarians an ideal choice for the study of regeneration. To develop a model in which to study the molecular basis of metazoan regeneration, the authors used the interference caused by double-stranded RNA (dsRNA) to eliminate gene expression in planarians. This approach has been shown in the past to specifically inhibit the expression of the gene from which the RNA was derived. The researchers injected dsRNA from body-wall myosin into the parenchyma or gastrovascular systems of planarians that had been cut transversely into 5-6 pieces. The results showed that injection of body-wall myosin inhibited the regeneration of body-wall musculature. They further showed that the site of dsRNA injection does not necessarily correspond to the site of its inhibitory action, suggesting the presence of a mechanism to deliver the dsRNA from extra- to intracellular compartments. Injection of opsin dsRNA followed by in situ hybridization was used to determine if the dsRNA interference acts by decreasing or eliminating endogenous mRNA transcripts. The results suggest that dsRNA injection interferes with gene expression by degrading the corresponding endogenous mRNA before translation. Promega's T3 (Cat.# P2083) and T7 (Cat.# P2075) RNA Polymerases were used to synthesize the dsRNA. Digoxigenin-labeled probes for in situ hybridization were prepared with the Riboprobe® System* (Cat.# P1420, P1430 and P1440).
Alvarado, A.S. and Newmark, P.A. (1999) Proc. Natl. Acad. Sci. USA 96, 5049.
Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210 USA.
The discovery of coelacanths off the coast of Manado Tua Island, Sulawesi, Indonesia, was reported in 1998. These coelacanths were the first recorded from a location outside of the western Indian Ocean and were caught almost 10,000km from the only other known population. The Indonesian fish was described as a new species, based on morphological differentiation and sequence divergence in fragments of the cytochrome b and 12S rRNA genes. These authors sequenced 4,823bp of mitochondrial DNA from the Indonesian coelacanth and found that the sequence was 4.1% different from the DNA of a coelacanth from the previously known Indian Ocean population, indicating substantial divergence between the two populations. The Wizard® PCR Preps DNA Purification System* (Cat.# A7170) was used to purify PCR(a) products amplified from coelacanth genomic DNA prior to sequencing.
Holder, M.T.1, Erdmann, M.V.2, Wilcox, T.P.1, Caldwell, R.L.2 and Hillis, D.M.1 (1999) Proc. Natl. Acad. Sci. USA 96, 12616.
1Section of Integrative Biology and Institute of Cellular and Molecular Biology, University of Texas, Austin, TX 78712; 2Department of Integrative Biology, University of California, Berkeley, CA 94720 USA.
The double subgenomic Sindbis (dsSIN) viruses, based on the mosquito-borne virus Sindbis (SIN; Togaviridae) allow stable, long-term cytoplasmic expression of genes of interest in mosquitoes. To determine whether dsSIN viruses could be used to target mRNAs transcribed from mosquito genes, these authors used a transgenic line of Aedes aegypti that expresses luciferase (luc). They hypothesized that the virus expression system may efficiently transcribe effector RNAs that inhibit expression of the targeted luciferase gene. Germ line transformed Ae. aegypti expressing luciferase were inoculated with dsSIN viruses that transcribed RNA complementary to the 5´ end of the luciferase mRNA or with control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with anti-luc viruses exhibited a 90% reduction in luciferase expression compared with controls, showing that a gene expressed from the mosquito genome can be inhibited using an antisense strategy. The Luciferase Assay System* (Cat.# E4030) was used to assay luciferase activity. The luciferase gene from the pGEM®-luc Vector* (Cat.# E1541) was used to construct the anti-luc dsSIN virus.
Johnson, B.W.1, Olson, K.E.1, Allen-Miura, T.1, Rayms-Keller, A.1, Carlson, J.O.1, Coates, C.J.2, Jasinskiene, N.3, James, A.A.3, Beaty, B.J.1 and Higgs, S.1 (1999) Proc. Natl. Acad. Sci. USA 96, 13399.
1Arthropod-Borne and Infectious Diseases Laboratory (AIDL), Department of Microbiology, Colorado State University, Fort Collins, CO 80523; 2Center for Advanced Invertebrate Molecular Sciences, Department of Entomology, Texas A&M University, College Station, TX 77843-2475; 3Department of Molecular Biology and Biochemistry, Bio Sci II, Room 3205, University of California, Irvine, CA 92697-3900 USA.
Synthetic peptides containing the arginine-glycine-aspartate (RGD) motif have been used extensively as inhibitors of integrin-ligand interactions in studies of cell adhesion, migration, growth and differentiation because the RGD motif is an integrin-recognition motif found in many ligands. These authors found that RGD-containing peptides were able to induce cell death directly in resting peripheral blood T cells, CD4-positive T cell lines, leukemic T cell lines (Jurkat and Molt-4 cells), B cells transformed by Epstein-Barr virus and the erythroleukemic cell line K562 without any requirement for integrin-mediated cell clustering or signals. Cell death was rapid, dose-dependent, and occurred over a low concentration range. RGD-containing peptides were demonstrated to enter cells and directly induce autoprocessing and activation of pro-caspase-3, a pro-apoptotic protein. In addition to an RGD motif, pro-caspase-3 contains a potential RGD-binding motif, aspartate-aspartate-methionine (DDM), near the site of processing to produce the p12 and p17 subunits. The authors suggest that RGD peptides induce apoptosis by triggering conformational changes that promote pro-caspase-3 autoprocessing and activation. These results challenge the prevailing paradigm that RGD peptides work only through effects on integrin-mediated cell adhesion and provide the first description of a peptide motif that is able to promote caspase-3 activation directly. The CaspACE™ Assay System, Fluorometric (Cat.# G3540) was used to measure caspase-1 and caspase-3 activity.
Buckley, C.D.1, Pilling, D.1, Henriquez, N.V.2, Parsonage, G.1, Thelfall, K.1, Scheel-Toellner, D.1, Simmons, D.L.3, Akbar, A.N.4, Lord, J.M.1 and Salmon, M.1 (1999) Nature 397, 534.
1Division of Immunity and Infection, and 2Institute for Cancer Studies, MRC Center for Immune Regulation, The University of Birmingham, Birmingham B15 2TT, UK; 3Department of Neuroscience SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK; 4Department of Clinical Immunology, Royal Free and University College Medical School, Pond Street, London NW3 2PF, UK.