CSM News
Electronic Edition
Volume 3, number 19
December 17, 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.

Back issues of CSM-News, the CSM Reference database and other useful
information is available by anonymous ftp from worms.cmsbio.nwu.edu
[165.124.233.50], via Gopher at the same address, or by World Wide Web
through www.nwu.edu.

---------------------
 Positions Available
---------------------

C1 position in Genetics/Molecular Biology available (postdoc, max. 5
years, possibility for habilitation) at the Dept. of Genetics,
University of Kassel, Germany,

This is a new laboratory which is currently being set up and should be
running by April/May 1995. Nevertheless, the successful applicant will
be required to participate in the initial establishment and
organization. The lab will be well equipped and sufficiently funded.
Applicants should be highly motivated and capable to establish a semi-
independent research project within the frame outlined below.
Application for extramural funding is strongly encouraged and could
provide resources for setting up a small group.  C1 positions require
some participation in teaching, therefore knowledge in German language
and/or commitment to learn German would be an advantage.  The
university is an equal opportunity employer and strongly encourages
the application of women.

Research projects:

1. Signal transduction/gene regulation in Dictyostelium 
   We are using REMI (restriction enzyme mediated integration) to
identify components of the signalling chains leading to regulation of
early developmental genes.

2. Mechanisms of antisense mediated gene silencing in Dictyostelium
   We are trying to identify potential components of the endogenous
antisense machinery (dsRNase, RNA binding proteins, etc.) and test
their function in antisense mechanisms.

3. Antisense mechanisms in plants
   In a new project we will extend the work on antisense mechanisms to
the plant field (probably tobacco and/or Arabidopsis) using our
knowledge (and material) from the Dictyostelium project as a basis.
References of recent papers can be provided upon request.

Interested individuals should contact me now by e-mail (address below)
providing a CV, names of two references and a brief summary of
research experience and the current work. I will pre-select candidates
and advice them when and where to send the official application. This
may seem complicated but is actually a shortcut in German burocracy!

wolfgang nellen
MPI Biochem.
D-82152 Martinsried
Germany
Tel. ++49 (89) 8578 2241
FAX  ++49 (89) 8578 3777
nellen@vms.biochem.mpg.de

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I have an immediate opening for a postdoctoral associate here at the
La Jolla Cancer Research Foundation.  There are several projects that
involve molecular aspects of Glycobiology in Dictyostelium.  These
include mostly investigation of phosphoglycosylation, a novel form of
glycosylation in Dicty.  We are trying to identify glycoyl transferase
genes, and to biochemically characterize these transferases as
well.A background in molecular aspects of Dicty is essential and some
knowledge of Glycobiology would be helpful. A competitive salary and
benefit package is available. Interested candidates should contact me
by e-mail, FAX or letter including a CV and the names of at least
three references.  Send by mail to : 

Hud Freeze, Senior Staff Scientist
La Jolla Cancer Research Foundation
10901 N. Torrey Pines Rd.
La Jolla CA 92037

PHONE: 619-455-6480


-----------
 Abstracts
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A protein kinase from Dictyostelium discoideum with an unusual acidic
repeat domain

B.W.Wetterauer1, U. Hamker, A. von Haeseler, H.K. MacWilliams, M.-N.
Simon and M. Veron

BBA, in press

We report the sequence of a protein kinase (DdKinX) from Dictyostelium
discoideum. It codes for a protein of 1093 amino acids, which are
organized in four regions (A) the N-terminal catalytic domain, (B) a
region containing 30% acidic amino acids, (C) a region which consists
of tandem repeats of the motif PVKVEEPVEE or variants of it and (D)
the C-terminus which is again rich in acidic amino acids and serine,
threonine and proline residues. The N-terminal sequence is in good
agreement with the consensus motifs for myristylation and the
C-terminus might be prenylated. DdKinX is weakly expressed. Similarity
to other protein kinases is low except for the short consensus motifs
(25 to 30% identity). Descendent trees calculated with the catalytic
core show that the branch leading to DdKinX evolved independently from
other kinase families. The root of the branch lies between mos and raf
serine-threonine kinases, tyrosine kinases and the other
serine-threonine kinases.

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Multiple Signal Transduction Pathways Regulate Discoidin I Gene
Expression in Dictyostelium discoideum

Jurgen Blusch1, Stephen Alexander2 and Wolfgang Nellen1

1 Max-Planck-Institut fur Biochemie, Abteilung Zellbiologie, D-82152,
Martinsried, Germany

2 Division of Biological Sciences, University of Missouri, Columbia,
Missouri 65211 USA

Differentiation, in press

Abstract.  

   The expression of the discoidin I genes in Dictyostelium discoideum
is regulated by the concerted action of the extracellular factors
cAMP, folate, PSF (Prestarvation Factor) and CMF (Conditioned Media
Factor).  However, the pathways by which these signals are transduced
and the interactions between the pathways were unexplored.  We have
analysed wild-type and mutant cells with defined lesions in signal
transduction to elucidate these regulatory processes.  We show that
different pathways are used for the down-regulation and induction of
these genes.  The cAMP receptor cARI is required for the cAMP mediated
down-regulation of discoidin I gene expression but not for the
induction of the expression during development.  Surprisingly, the
induction of the discoidin I genes requires Ga2, the G-protein subunit
which is generally believed to couple to cARI, to control the
expression of cAMP inducible genes.  Thus, our data suggests that Ga2
interacts with different receptors to regulate gene expression in
early development.  Furthermore, the analysis shows that discoidin
induction in bacterially grown cells occurs in two sequential steps.
The first is a low basal induction which occurs in late-log phase
growth prior to starvation.  PSF can induce the basal level and the
induction is independent of Ga2.  The developmental induction
following starvation is much stronger, dependent on Ga2 and probably
signaled by CMF which is secreted at this time.  We also demonstrate
that the growth history of the cells has a major influence on the
pattern of gene expression and on the choice of the signal
transduction pathway that is used to induce the discoidin I genes.
Overall, these studies reveal complex regulatory interactions between
the stimulatory and inhibitory signaling pathways controlling the
expression of the discoidin I gene family.

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Growth and developmental functions of a human HIV-Tat binding protein/26S
protease subunit homologue from Dictyostelium  discoideum

Jie-Gang Cao and Richard A Firtel

Department of Biology, Center for Molecular Genetics, University of
California, San Diego, La Jolla, California 92093-0634. telephone:
619-534-2788, fax: 619-534-7073

Mol. Cell. Biol., in press.

ABSTRACT

   We have characterized a newly identified gene from Dictyostelium,
DdTBPa, that encodes a member of the family of eukaryotic proteins
containing a conserved ATPase domain that include subunits of the 26S
protease subunit and are homologous to the mammalian HIV-Tat binding
protein TBP1.  While information indicates that some family members
are involved in regulating transcription during growth in mammalian
cells and yeast, these proteins are also involved in other cellular
functions, and nothing is known about their possible function in
multicellular development.  The Dictyostelium DdTBPa gene is
developmentally regulated, being expressed at the highest levels
during growth and early development.  The gene is present in two
copies in the genome.  Disruption of one copy by homologous
recombination leads to aberrant morphogenesis from the formation of
the first finger until the onset of culmination.  The gene appears to
be essential for growth since we were unable to obtain a complete null
phenotype and expression of an inducible antisense construct in the
partial null resulted in cell death.  Expression of the antisense
construct during development accentuated the partial null phenotype
and also resulted in very abnormal fruiting bodies.  Overexpression of
DdTBPa from its own promoter leads to very large multinucleated
vegetative cells when the cells are grown in suspension culture.  When
plated onto Petri dishes in growth medium, the cells rapidly split
into multiple cells containing 1-2 nuclei, similar to wild-type cells.
Overexpressing cells are significantly delayed in forming a
multicellular aggregate, but development proceeds normally once the
first finger stage is reached.  The results indicate that DdTBPa plays
an important role in regulating both growth and morphogenesis in
Dictyostelium.

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Glycoprotein complexes interacting with cellulose in the "cell print"
zones of the Dictyostelium discoideum extracellular matrix.

Ti Zhou-Chou, Wilkins, M.R., Vardy, P.H., Gooley, A.A. & Williams,
K.L. 

Developmental Biology, in press

Abstract

   Cellulose is one of the commonest structural biopolymers.  How
cellulose is organized in extracellular matrices is a mystery.  Here
we investigate a model system, the extracellular matrix (ECM) of
Dictyostelium discoideum which is composed of proteins and cellulose.
A group of glycoproteins, the sheathins, which co-localize with
cellulose in the ECM of D. discoideum are characterized.  Sheathins
are dimeric or trimeric forms of molecular mass 53-68 kDa; where the
monomers are 12-35 kDa.  The sheathin subunits are similar but not
identifical proteins.  The sheathin family comprises sheathin 68 (68
kDa trimer); sheathin 62, kDa dimer); sheathin 55,(55kDa dimer) and
sheathin 53,(53kDa dimer).  The subunits which assemble into the four
sheathins represent at least three gene products: ShC, ShD and ShE
which are linked by disulphide bonds.  Protein sequence analysis shows
two of the sheathin genes encode products ShC and ShD with very
similar amino-terminal sequences.  This group of D.discoideum ECM
glycoproteins has homology with two other much larger ECM proteins of
D.discoideum, ST430 and ST310, which are located in a more dispersed
fashion in the ECM.  Sheathins are tightly but non-covalently
associated with the ECM, and this association requires strong
denaturing conditions for disruption, eg. SDS or 8 M urea.  Sheathins
from a compnent of the "cell prints" which are believed to have a role
in cell- ECM interations and slug cell migration.

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Immunochemical, Genetic and Morphological Comparison of Fucosylation
Mutants of Dictyostelium discoideum 

Champion, A.,  Griffiths, K., Gooley, A.A., Gonzalez, B.Y., Gritzali, M.,
West, C.M. & Williams, K.L. 

Abstract

   Mutations in three loci in Dictyostelium discoideum which affect
fucosylation are described; mutations in two of these loci result in
the simultaneous loss of two separate carbohydrate epitopes.  The Ga-X
epitope, which is competed by L-fucose, is absent in strains carrying
a modC354, modD352 or modE353 mutation.  These strains expose a new
carbohydrate epitope, competed by N-acetylglucosamine, and the size of
several glycoproteins is reduced.  A second epitope (GA-XII) is also
absent in strains carrying the modC354 or mod E353 mutations, reducing
the size of the glycoprotein which normally expresses it.  Fucose
content is reduced in the three mutants, suggesting that each mutation
affects a separate step in fucosylation.  The lesions do not appear to
inhibit synthesis of the underlying carbohydrate, because detergent
extracts of mutant vesicles are more active than normal vesicles at
transferring (14C) fucose from GDP-(14C) fucose to endogenous acceptor
species.  The modD352 and modE353 mutant strains incorporate exogenous
(3H) fucose poorly, suggesting that lesions in the modD and modE genes
interfere with the biosynthesis of fucoconjugates downstream from the
previously described GDP-fucose synthesis defect of the modC mutation.
Intact modE353 mutant vesicles are relatively inefficient in in vitro
assays, suggesting a global fucosylation defect (which is consistent
with the loss of both glycoantigens CGA-X and GA-XII in this mutant).
Finally, the modC354 mutation leads to delayed accumulation of slime
sheath in vitro.  The three genetic loci define a fucosylation pathway
in D.discoideum consisting of defined biochemical steps which
contribute to multicellular morphogenesis in this organism.

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The pH-sensitive actin-binding protein hisactophilin of Dictyostelium
exists in two isoforms which both are myristylated and distributed
between plasma membrane and cytoplasm

Frank Hanakam, Christoph Eckerskorn, Friedrich Lottspeich, Annette
Muller-Taubenberger, Wolfram Schufer, and Gunther Gerisch

Journal Biol. Chem., in press

Abstract

   The histidine-rich protein hisactophilin is known to be associated
with the inner surface of the plasma membrane and to be present as a
soluble protein in the cytoplasm of Dictyostelium discoidem cells. On
the basis of mass spectrometric data, hisactophilin from the cytosol
was analysed in parallel with hisactophilin extracted from a membrane
fraction. None of the hisactophilin purified from D. discoideum cells
had the mass predicted from the known cDNA-derived amino-acid sequence
of the protein, suggesting posttranslational modification. ES-MS and
GC-MS of tryptic fragments separated by reversed-phase HPLC identified
the most hydrophobic peptide as a myristylated fragment from the
N-terminus of hisactophilin. From combination of the analytical data
obtained, it is concluded that all hisactophilin in D. discoideum
cells is N-terminally modified by myristylation. By reversed-phase
HPLC, the D. discoideum hisactophilin could be separated into two
isoforms which were recovered from both the cytosolic and membrane
fraction. These isoforms, Hs I and Hs II, are distinguished by
amino-acid sequence. Whereas the masses of Hs I fragments produced by
trypsin fit into the previously published sequence of hisactophilin
(myristylation considered), Hs II is another protein distinguished
from Hs I by several amino-acid exchanges. Hs I and Hs II can form
homo- and heterodimers by disulfide bridges. Both isoforms proved to
be substrates of membrane-associated threonine/serine kinase from D.
discoidem, which may regulate the interaction of hisactophilin with
the plasma membrane.

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[End CSM News, volume 3, number 19]