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dictyNews Volume 22 Number 10
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.
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Abstracts
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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]
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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]
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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]
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[End Dicty News, volume 22, number 10]
