CSM News
Electronic Edition
Volume 6, number 5
February 24, 1996

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

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at the URL "http://worms.cmb.nwu.edu/dicty.html"

===========
 Abstracts
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Mutagenesis and gene identification in Dictyostelium by shotgun
antisense

Timothy P. Spann, Debra A. Brock, David F. Lindsey, Salli A. Wood, and
Richard H. Gomer

Howard Hughes Medical Institute and Department of Biochemistry and
Cell Biology, Rice University, Houston, TX 77251-1892

PNAS, in press

ABSTRACT

   We have developed a mutagenesis technique that uses antisense cDNA
to identify genes required for development in Dictyostelium
discoideum.  We transformed Dictyostelium cells with a cDNA library
made from the mRNA of vegetative and developing cells.  The cDNA was
cloned in an antisense orientation immediately downstream of a
vegetative promoter, so that in transformed cells the promoter will
drive the synthesis of an antisense RNA transcript.  We find that
individual transformants typically contain one or occasionally two
antisense cDNAs.  Using this mutagenesis technique, we have generated
mutants which either fail to aggregate, aggregate but fail to form
fruiting bodies, or aggregate but form abnormal fruiting bodies.  The
individual cDNA molecules from the mutants were identified and cloned
using PCR.  Initial sequence analysis of the PCR products from 35
mutants has identified six novel Dictyostelium genes, each from a
transformant with one antisense cDNA.  When the PCR-isolated antisense
cDNAs were ligated into the antisense vector and the resulting
constructs transformed into cells, the phenotypes of the transformed
cells matched those of the original mutants from which each cDNA was
obtained.  We made homologous recombination gene disruption
transformants for three of the novel genes, in each case generating
mutants with phenotypes indistinguishable from those of the original
antisense transformants.  Shotgun antisense thus is a rapid way to
identify genes in Dictyostelium and possibly other organisms.

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Cyclic AMP dependent protein kinase and prestalk-cell gene expression
in Dictyostelium.

Fabiana Horn and Julian D. Gross

Department of Biochemistry, University of Oxford
South Parks Road,Oxford OX1 3QU, U.K.

FEMS Microbiol. Letters, in press.

ABSTRACT

   Cyclic AMP-dependent protein kinase (PKA) is required for
aggregation of starved amoebae during development of Dictyostelium
discoideum as well as for prespore gene expression and for maturation
of stalk cells and spores. It has not been clear until now whether it
plays any role in the stalk pathway prior to the final maturation of
stalk cells. We have examined stalk cell formation and prestalk cell
gene expression in a mutant of Dictyostelium that has an inactive PKA
regulatory subunit and therefore has unrestrained catalytic activity
[1]. We show that when mutant amoebae are incubated under buffer in
the absence of cAMP they accumulate transcripts of the
prestalk-cell-specific ecmA gene and form stalk cells, whereas the
parental cells neither accumulate ecmA transcripts nor form stalk
cells. These findings indicate that constitutive PKA activity renders
cells able to express prestalk-cell-specific genes under conditions
where wild type cells cannot do so, and hence that PKA is probably
implicated in prestalk gene expression during normal development.

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MULTIPLE ROLES OF THE NOVEL PROTEIN-TYROSINE PHOSPHATASE PTP3 DURING
DICTYOSTELIUM GROWTH AND DEVELOPMENT

Marianne Gamper, Peter K. Howard, Tony Hunter, and Richard A. Firtel

Molecular and Cellular Biology, in press.

ABSTRACT

   PTP3, the third nonreceptor protein tyrosine phosphatase identified
in Dictyostelium discoideum, has a single catalytic PTP domain.
Recombinant PTP3 exhibited phosphatase activity that was inhibited by
vanadate.  PTP3 is expressed at a moderate level during growth.  The
level of transcripts increased between growth and 8 h of development
and declined thereafter.  Expression of lacZ under the control of the
PTP3 promoter indicated a spatial localization of PTP3 in the
anterior-like and prestalk cell-types.  There are two copies of the
PTP3 gene in this haploid organism.  Disruption of one copy led to a
slow growth phenotype.  We were unable to obtain a strain with
disruptions in both PTP3 genes.  Overexpression of wild-type PTP3 led
to slower growth rates and the formation of large aggregation streams.
These streams split into smaller aggregates, many of which then
arrested in development.  Overexpression of a catalytically inactive
mutation (Cys->Ser) had no effect on growth rate; however, this strain
also formed large aggregation streams that later split up into large
and small mound structures and became fruiting bodies of variable
size.  Anti-phosphotyrosine (pTyr) Western blot analysis of total cell
proteins showed that the pattern of protein tyrosine phosphorylation
was specifically altered in PTP3 mutants.  Addition of growth medium
to starving cells and a subsequent replacement with non-nutrient
buffer led to reciprocal changes in the pattern of several pTyr
proteins, including a protein of ~130 kDa.  Analysis of strains
overexpressing active or inactive PTP3 suggested p130 is a potential
substrate of PTP3.  A transient post-translational phosphorylation of
PTP3 further supported the role of PTP3 in these processes.  The data
shown strongly suggest new regulatory functions for PTP3 that are
distinct from those described earlier for D.  discoideum PTP1 and
PTP2.

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MOLECULAR GENETICS OF SIGANL TRANSDUCTION IN DICTYOSTELIUM

Carole A. Parent and Peter N. Devreotes

Annual Review of Biochemistry, in press.

ABSTRACT

   In conditions of starvation, the free living amoebae of
Dictyostelium enter a developmental program - the cells aggregate by
chemotaxis to form a multicellular structure that undergoes
morphogenesis and cell-type differentiation. These processes are
mediated by a family of cell surface cAMP receptors (cARs) that act on
a specific heterotrimeric G protein to stimulate actin polymerization,
activation of adenylyl and guanylyl cyclases, and a host of other
responses. Most of the components in these pathways have mammalian
counterparts. The accessible genetics of this unicellular organism
facilitate structure-function analysis and enable the discovery of
novel genes involved in the regulation of these important pathways.

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Evidence that a combined activator-repressor protein regulates
Dictyostelium stalk cell differentiation

Takefumi Kawata*, Anne Early* and Jeffrey Williams+

MRC Laboratory of Molecular Cell Biology and Dept of Biology
University College London, Gower St, London WC1E 6BT, U.K.

*Joint first authors

EMBO J., in press.
                              
ABSTRACT

   The ecmA gene is expressed in Dictyostelium prestalk cells and is
inducible by DIF, a low molecular weight lipophilic substance. The
ecmB gene is expressed in stalk cells and is under negative control by
two repressor elements. Each repressor element contains two copies of
the sequence TTGA in an inverted relative orientation. There are
activator elements in the ecmA promoter that also contain two TTGA
sequences but in the same relative orientation. Gel retardation assays
suggest that the same protein binds to the ecmB repressor and the ecmA
activator. We propose that DIF induces prestalk cell differentiation
by activating this protein and that the protein also binds to the
promoters of stalk-specific genes, acting as a repressor that holds
cells in the prestalk state until culmination is triggered.

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Dimer Formation of a Cell-Cell Adhesion Protein, gp64 of the Cellular
Slime Mold, Polysphondylium pallidum

Ochiai Hiroshi, Hata Katsusuke, Saito Tamao, Funamoto Satoru, and
Nakata Nobuyuki

Division of Biological Sciences, Graduate School of Science, Hokkaido
University, Sapporo, 060 Japan

Plant Cell Physiology, in press.

SUMMARY

   The cellular slime mold, Polysphondylium pallidum, has two
EDTA-resistant types of cell-cell adhesion.  The major component of
them has been identified as a glycoprotein with a molecular mass of 64
kDa on SDS-PAGE (referred to as gp64).  We found that a substantial
amount of the gp64 run as dimer, when gp64 was dissolved in SDS-sample
buffer without 2-mercaptoethanol and then subjected to
electrophoresis.  The occurrence of a homophilic dimer was
demonstrated by analyzing the dimer-like band on a gel for its amino
acid sequence and amino acid composition.  The dimer-like band also
was analyzed by three sorts of monoclonal antibodies, two of which
recognize respectively a conformational epitope and a denatured
epitope of the protein moiety of gp64.  The data indicate that the
native conformation of gp64 is necessary for dimer formation.  The
cellular slime mold Polysphondylium pallidum is like a related
species, Dictyostelium discoideum, whose cells migrate in response to
a chemoattractant secreted from center cells and finally form a
multicellular aggregate (Gerisch et al. 1980).  When cells of the two
species are mixed and plated onto a solid agar surface, they sort out
into separate aggregates not only because of differences in
chemoattractants but also because of species-specific cell-cell
adhesion (Gerisch et al. 1980).  D.  discoideum has two types of
adhesion systems, named the contact site A and the contact site B
(Beug et al. 1973).  The contact site A was identified as a
glycoprotein with a molecular mass of 80 kDa (Mueller et al ., 1979)
and has been well studied.  Its cDNA sequence (Noegel et al., 1986),
an adhesion site (Kamboj et al. 1989) and a glycosyl
phosphatidylinositol anchor (Stadler et al. 1989) have been reported.
The contact site B also has been preliminary identified as a gp120
(Chadwick et al. 1984), or a gp 24 (Knecht et al. 1987).  In the
related species P. pallidum two EDTA-resistant types of cell adhesion
have been described (Bozzaro and Gerisch, 1978).  One is a minor
component which begins to appear at aggregation stage and the other is
a major component expressed already during growth phase and persisting
until later stages.  The major component was identified as s
glycoprotein with a molecular mass of 64 kDa on SDS-PAGE (referred to
as gp64) (Bozzaro et al. 1981).  Monoclonal antibodies against
purified gp64 completely blocked cell-cell adhesion of
aggregation-competent P. pallidum cells and this blocking activity was
neutralized with L-fucose (Toda et al 1984).  This result indicates
that the carbohydrate moiety of gp64 is involved in cell-cell
adhesion.  Recently, by subjecting purified gp64 to electrophoresis,
we recognized the presence of a dimer-like molecule on SDS-gel.
Bozzaro et al. (1981) also noticed the same thing, but they did not
analyze the nature of the dimer though they suggested that gp64 has a
tendency to dimerize.  We show here that the dimer-like product is a
homophilic dimer of gp64 molecules and that the monomers might have a
rather strong affinity to interact to each other.

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Antisense RNA Inactivation of Gene Expression of a Cell-Cell Adhesion
Protein (gp64) in the cellular slime mold Polysphondylium pallidum

Satoru Funamoto and Hiroshi Ochiai

Division of Biological Sciences, Graduate School of Science, Hokkaido
University, Sapporo, 060 Japan

J. Cell Sci. in press

SUMMARY

   The gp64 protein of Polysphondylium pallidum has been shown to
mediate EDTA-stable cell-cell adhesion.  To explore the functional
role of gp64, we made an antisense RNA expression construct designed
to prevent the gene expression of gp64; the construct was introduced
into the cells, and the resultant was characterised.  The antisense
RNA-expressing clone L3mc2 which had just been harvested at the growth
phase tended to re-form in aggregates smaller in size than the
parental cells in either the presence or absence of 10 mM EDTA.  In
contrast, 6.5-hours starved L3mc2 cells remained considerably
dissociated from each other after 5-minutes gyrating, although then
gradually increased aggregation by 50% during a further 55-minutes
gyrating, in the presence of 10 mM EDTA.  Correspondingly, L3mc2
lacked specifically the cell-cell adhesion protein, gp64.  We
therefore conclude that gp64 protein is involved in forming the
EDTA-resistant cell-cell contact.  In spite of the absence of gp64,
L3mc2 exhibited normal developmental processes, a fact which
demonstrates that another cell-cell adhesion system exists in the
development of Polysphondylium.  This is the first report which an
antisense RNA technique was successfully applied to Polysphondylium.

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Identification of a novel all-cis-5,9,12-heptadecatrienoic acid in the
cellular slime mold Polysphondylium pallidum

Tamao Saito and Hiroshi Ochiai

Division of Biological Sciences, Graduate School of Science, Hokkaido
University, Sapporo, 060 Japan

Lipids, in press.

Summary

   All-cis-5,9,12-heptadecatrienoic acid, was identified in the
cellular slime mold Polysphondylium pallidum.  The structural
elucidation was accomplished by capillary GC, argentation TLC and
GC/MS.  This fatty acid has not been reported previously.


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[End CSM News, volume 6, number 5]