CSM News
Electronic Edition
Volume 3, number 7
September 3, 1994

Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu.

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through www.nwu.edu.

===========
 Abstracts
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Temesvari, L.A., Rodriguez-Paris, J., Bush, J., Steck, T.L., and
Cardelli, J.  

Characterization of Lysosomal Membrane Proteins of Dictyostelium
discoideum: A Complex Population of Acidic Integral Membrane
Glycoproteins, Rab GTP-binding Proteins and Vacuolar ATPase Subunits.

J. Biol. Chem., in press.

Abstract

   Highly purified lysosomes, prepared by magnetic fractionation of
homogenates from Dictyostelium discoideum cells fed colloidal iron,
were lysed under hypo-osmotic conditions, and the membrane-associated
proteins were subjected to gel electrophoresis.  Thirteen major
membrane polypeptides, ranging in molecular weight from 25 kd to 100
kD were identified.  The isoelectric points of these proteins ranged
from below 3.8 to greater than 7.0.  Most of these proteins were
stripped from membranes exposed to a chaotropic agent,
3,5,-diiodo-2-hydroxybenzoic acid lithium salt, and were therefore
classified as peripheral membrane proteins.  Twenty five glycoprotein
species were detected by lectin blot analysis; 19 were classifed as
integral membrane proteins, and were in general, larger than 45 kD and
negatively charged due in part to the presence of mannose-6-sulphate.
Western blot analysis also demonstrated that a Rab 4-like GTPase, a
Rab 7-like GTPase and at least three subunits of the vacuolar ATPase
were associated with the lysosomal membrane; the ATPase subunits
appeared to be major proteins in lysosomal membranes.  Finally, based
on N-terminal sequence analysis of a major 41 kD lysosome-associated
membrane proteins, we cloned a cDNA that encodes a protein (DVA41)
highly homolgous to a yeast and a bovine vacuolar ATPase subunit of
approximately 41 kD.  The D. discoideum DVA41 gene was apparently a
single copy gene, expressed at constant levels during growth and
development.

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ANALYSIS OF SUCCESSIVE ENDOCYTIC COMPARTMENTS ISOLATED FROM
DICTYOSTELIUM DISCOIDEUM BY MAGNETIC FRACTIONATION

Kathleen V. Nolta, Juan M. Rodriguez-Paris#, Theodore L. Steck*

Department of Biochemistry and Molecular Biology The University of
Chicago 920 East 58th Street, Chicago, IL 60637

*Corresponding author.  Phone: (312) 702-1329;
                        FAX: (312) 702-0439
                        E-mail: t-steck@uchicago.edu

#Present address: Dept. of Microbiology and
Immunology, Louisiana State University Medical Center,
1501 Kings Highway, Shreveport, LA 71130

Biochimica et Biophysica Acta, in press

Abstract  

   A colloidal iron probe was fed to the amoeba, Dictyostelium
discoideum, and chased for different intervals.  Successive segments
of the endocytic pathway were then isolated magnetically at high yield
and purity.  There were ~500 endocytic vacuoles per cell; their
diameters increased from ~0.1-0.2 um after 3 min of feeding to ~2 um
after 15 min of feeding and 60 min of chase.  The wave-like
progression of ingested probes along the endocytic pathway suggested
that the transfer of cargo involved a maturation mechanism rather than
the shuttling of cargo between stable compartments.  The lifetime of
primary pinosomes was calculated to be ~1 sec.  Multivesicular bodies
were common in the 3-min fraction and abundant in 15 min lysosomes.

     Alpha- and beta-adaptins of Mr ~89 and ~83 kDa were richer in the
3-min vesicles than in plasma membranes and later endocytic vacuoles.
Acid phosphatase, intrinsic vacuole acidity, the vacuolar proton pump
protein and pump activity were present at all endocytic stages but
rose between the 3 min and 15 min vacuoles and declined thereafter.
Bis(monoacylglycero)phosphate or BMP, a lipid characteristic of
lysosomes, followed a similar time course; it contributed up to half
of the total lipid in lysosomal vacuoles. We conclude that there is
both continuity and differentiation along this endocytic pathway.

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Developmental regulation of antisense mediated gene silencing in
Dictyostelium.

May Sadiq#, Martin Hildebrandt$, Markus Maniak and Wolfgang Nellen*

Max-Planck-Institut f. Biochemie, D-82152 Martinsried, Germany
# Yarmouk University, Irbid, Jordan
$ Inst. Pasteur, Paris, France
* corresponding author

Antisense Research & Development, in press.

Abstract

   In Dictyostelium, the expression of antisense transcripts has been
successfully used to reduce or eliminate gene expression. In most
cases this occurs on the level of RNA stability resulting in a loss of
both sense and antisense transcript accumulation. We here show that
the antisense effect is regulated during the developmental cycle, i.e.
in certain developmental stages and under certain developmental
conditions, complementary RNAs appear not to interact with each other,
resulting in a failure to abolish expression of the gene of interest.
We find that this is not only the case with artificially introduced
antisense constructs but also with the endogenous, antisense regulated
PSV-A gene. Our data demonstrate that antisense mediated gene
silencing is conferred by a biochemical machinery which is subject to
regulation in vivo. The results provide a basis to better understand
this machinery and to dissect the components.  They may also explain
the failure of some antisense experiments in Dictyostelium and
possibly in other organisms.

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[End CSM-News, volume 3, number 7]