Dicty News Electronic Edition Volume 22, number 10 April 30, 2004 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Preparation of genomic DNA from Dictyostelium discoideum for PCR analysis. Charette S.J. and Cosson P. Universite de Geneve, Centre Medical Universitaire, Departement de Physiologie Cellulaire et Metabolisme, 1 rue Michel Servet, CH-1211 Geneve 4, Switzerland. Biotechniques. 2004 Apr;36(4):574-5. In this paper, we present a very quick protocol (less than 5 minutes) to prepare genomic DNA from D. discoideum cells suitable for PCR analysis. This approach facilitates the screening of resistant clones to identify those with the selection cassette in the desired gene. The method works for cells grown in liquid culture or on agar plates. Genomic DNA produced by this method can be also used as template for cloning or for any other PCR based applications. A summary of the protocol will be available on DictyBase (http://dictybase.org/techniques/quick_gDNA.htm). Submitted by: Steve Charette [charett2@etu.unige.ch] ----------------------------------------------------------------------------- Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways Christopher M. West1, Hanke van der Wel1, Slim Sassi2, Eric A. Gaucher3 1 Dept. of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104 USA; 2 Dept. of Anatomy & Cell Biology, University of Florida, Gainesville, FL, 32610 USA; and 3 Foundation for Applied Molecular Evolution, Gainesville, FL, 32601 USA Biochim. Biophys. Acta, in press The Skp1 protein, best-known as a subunit of E3SCF-ubiquitin ligases, is subject to complex glycosylation in the cytoplasm of the cellular slime mold Dictyostelium. Pro143 of this protein is sequentially modified by a prolyl hydroxylase and five soluble glycosyltransferases, to yield the structure Gala1,Gala1,3Fuca1,2Galb1,3GlcNAca1-HyPro143. These enzymes are unusual in that they are expressed in the cytoplasmic compartment of the cell, rather than the secretory pathway where complex glycosylation of proteins usually occurs. The first enzyme in the pathway appears to be related to the soluble animal prolyl 4-hydroxylases that modify the transcriptional factor subunit HIF-1a in the cytoplasm, and more distantly to the prolyl 4-hydroxylases that modify collagen and other proteins in the rER, based on biochemical and informatics analyses. The soluble aGlcNAc-transferase acting on Skp1 has been cloned and is distantly related to the mucin-type polypeptide aGalNAc-transferases in the Golgi of animals. Its characterization has led to the discovery of a family of related polypeptide aGlcNAc-transferasesin the Golgi of selected lower eukaryotes. The Skp1 GlcNAc is extended by a bifunctional diglycosyltransferase that sequentially and apparently processively adds b1,3Gal and a1,2Fuc. Though this structure is also formed in the animal secretory pathway, the glycosyltransferases involved are dissimilar. Conceptual translation of available genomes suggests the existence of this kind of complex cytoplasmic glycosylation in other eukaryotic microorganisms, including diatoms, oomycetes, and possibly Chlamydomonas and Toxoplasma, and an evolutionary precursor of this pathway may also occur in prokaryotes. Submitted by: Chris West [Christopher-West@ouhsc.edu] ----------------------------------------------------------------------------- Talin B is required for force transmission in morphogenesis of Dictyostelium Masatsune Tsujioka(1), Kunito Yoshida(2) and Kei Inouye(3) Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. (1)Present address: Gene Function Research Center, Tsukuba Central 4, AIST, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan (2)Present address: Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, England (3)Correspondence: inoue@cosmos.bot.kyoto-u.ac.jp EMBO J., in press Talin plays a key role in the assembly and stabilisation of focal adhesions, but whether it is directly involved in force transmission during morphogenesis remains to be elucidated. We show that the traction force of Dictyostelium cells mutant for one of its two talin genes talB is considerably smaller than that of wild-type cells, both in isolation and within tissues undergoing morphogenetic movement. The motility of mutant cells in tightly packed tissues in vivo or under strong resistance conditions in vitro was lower than that of wild-type cells, but their motility under low external force conditions was not impaired, indicating inefficient transmission of force in mutant cells. Antibody staining revealed that the talB gene product (talin B) exists as small units subjacent to the cell membrane at adhesion sites without forming large focal adhesion-like assemblies. The total amount of talin B on the cell membrane was larger in prestalk cells, which exert larger force than prespore cells during morphogenesis. We conclude that talin B is involved in force transmission between the cytoskeleton and cell exterior. Submitted by: K. Inouye [inoue@cosmos.bot.kyoto-u.ac.jp] =============================================================================== [End Dicty News, volume 22, number 10]