CSM News
Electronic Edition
Volume 7, number 15
December 14, 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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information is available by anonymous ftp from worms.cmb.nwu.edu
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at the URL "http://worms.cmb.nwu.edu/dicty.html"

===========
 Abstracts
===========


Substitution Mutations in the Myosin Essential Light Chain Lead to
Reduced Actin-activated ATPase Activity Despite Stoichiometric Binding
to the Heavy Chain

Guyu Ho and Rex L. Chisholm*

Department of Cell and Molecular Biology, Northwestern University
Medical School, Chicago, Illinois 60611

J. Biol. Chem.,  in press.

   Myosin essential light chain (ELC) wraps around an alpha-helix that
extends from the myosin head, where it is believed to play a
structural support role. To identify other role(s) of the ELC in
myosin function, we have used an alanine scanning mutagenesis approach
to convert charged residues in loops I, II, III, and helix G of the
Dictyostelium ELC into uncharged alanines. Dictyostelium was used as a
host system to study the phenotypic and biochemical consequences
associated with the mutations. The ELC carrying loop mutations bound
with normal stoichiometry to the myosin heavy chain when expressed in
ELC-minus cells. When expressed in wild-type cells these mutants
efficiently competed with the endogenous ELC for binding, suggesting
that the affinity of their interaction with the heavy chain is
comparable to wild-type. However, despite apparently normal
association of ELC the cells still exhibited a reduced efficiency to
undergo cytokinesis in suspension.  Myosin purified from these cells
exhibited four to five fold reduction in actin-activated ATPase
activity and a decrease in motor function as assessed by an in vitro
motility assay. These results suggest that the ELC contributes to
myosin's enzymatic activity in addition to providing structural
support for the *-helical neck region of myosin heavy chain.

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Relevance of Histone H1 Kinase Activity to the G2/M Transition during
the Cell Cycle of Dictyostelium discoideum

Tohru Arakane and Yasuo Maeda*

Biological Institute, Graduate School of Science, Tohoku University,
Aoba, Sendai, 980-77 Japan

J. Plant Res., in press.   

   The implication of histone H1 kinase activity for the G2/M
transition during the cell cycle was investigated using Dictyostelium
discoideum Ax-2.  Histone H1 kinase with its activity was purified
from cell extracts by the use of p13suc1 affinity gel. In the
vegetative cell cycle, the activity of histone H1 kinase including
Cdc2 kinase was found using synchronized Ax-2 cells to be highest just
before the entry into mitosis. The activity also was markedly enhanced
just prior to the M phase from which developing cells (possibly
prespore cells ) reinitiate their cell cycle at the mound-tipped
aggregate stage. These results strongly suggest the importance of Cdc2
kinase activity in the G2 to M phase transition during the cell cycle,
as the case for other eukaryotic cells.

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Group I introns in the cytochrome oxidase genes of Dictyostelium
discoideum: two related ORFs in one loop of a group I intron, cox1/2
hybrid gene and increased-size cox3 gene

Shinji Ogawa, Kuniko Matsuo, Kiyohiko Angata, Kaichiro Yanagisawa and
Yoshimasa Tanaka*

Institute of Biological Sciences and Center for TARA*, University of
Tsukuba, Tsukuba, Ibaraki 305, Japan

Curr. Genet., in press

   The DNA sequences of cytochrome oxidase (subunits 1, 2 and 3) genes
of the cellular slime mold Dictyostelium discoideum mitochondria were
determined.  The genes for subunits 1 and 2 have a single continuous
ORF (COX1/2) which contains four group-I introns.  The insertion sites
of the two group I introns (DdOX1/2.2 and DdOX1/2.3) coincide with
those of fungal and algal as well as a liverwort group-I intron, in
the cytochrome oxidase subunit 1.  Interestingly, intron DdOX1/2.2 has
two free-standing ORFs in a loop (L8) which have similar amino-acid
sequences and are homologous to ai4 DNA endonuclease (I-Sce II) and
bi4 RNA maturase found in group-I introns of Saccharomyces cerevisiae
mitochondrial DNA.  Two group-I introns (DdOX1/2.3 and DdOX1/2.4) also
have a free-standing ORF in loop 1 and loop 2, respectively.  These
results show that these group-I introns and the intronic ORFs have
evolved from the same ancestral origin, but that these ORFs have been
propagated independently.

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The Dictyostelium MAP Kinase ERK2 is Regulated by Ras and
cAMP-Dependent Protein Kinase (PKA) and Mediates PKA Function

Laurence Aubry*, Mineko Maeda*, Robert Insall, Peter N. Devreotes, and
Richard A. Firtel *These two authors contributed equally to this
manuscript.

J. Biol. Chem., in press

   The chemoattractant cAMP, acting through serpentine cAMP receptors,
results in a rapid and transient stimulation of the Dictyostelium MAP
kinase ERK2 activity (1) .  In this study we show that other pathways
required for aggregation, including Ras and cAMP-dependent protein
kinase (PKA), are important regulators of ERK2 activation and
adaptation.  By examining both the level and kinetics of activation
and adaptation of ERK2, we show that Ras is a negative regulator of
ERK2.  Cells expressing an activated Ras protein or mutant in a Ras
GAP demonstrate a reduced ERK2 activation profile while cells mutant
in a putative RasGEF or expressing dominant negative Ras proteins have
a more rapid, higher, and extended activation profile.  We show that
CRAC, a PH domain-containing protein required for the activation of
adenylyl cyclase, is also required for proper adaptation of ERK2.
Overexpression of PKA results in a more rapid and higher level of
activation, while cells in which the PKA catalytic subunit has been
disrupted show a slightly lower maximal level of activation, and more
extended kinetics.  Furthermore, we show that constitutive expression
of PKA catalytic subunit bypasses the requirement of ERK2 for
aggregation and later development.  This indicates that PKA lies
downstream from ERK2 and suggests that ERK2 may regulate one or more
component of the signaling pathway required for mediating PKA
function, possibly by directly regulating PKA R or C subunits or
another protein controlling the intracellular level of cAMP.  These
data provide new insights into pathways by which classic G
protein-coupled chemoattractant receptors regulate MAP kinase cascades
and the regulatory loops controlling aggregation in Dictyostelium.

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A Ras GAP is essential for cytokinesis and spatial patterning in
Dictyostelium

Susan Lee, Ricardo Escalante, and Richard A. Firtel

DEVELOPMENT, in press

   Using the yeast two-hybrid system, we have identified
developmentally regulated Dictyostelium genes whose encoded proteins
interact with Ras.GTP but not Ras.GDP.  By sequence homology and
biochemical function, one of these genes encodes a Ras GAP
(DdRasGAP1).  Cells carrying a DdRasGAP1 gene disruption (ddrasgap1
null cells) have multiple, very distinct growth and developmental
defects as elucidated by examining the phenotypes of ddrasgap1 null
strains.  First, vegetative ddrasgap1 null cells are very large and
highly multinucleate cells when grown in suspension, indicating a
severe defect in cytokinesis.  When suspension-grown cells are plated
in growth medium on plastic where they attach and can move, the cells
rapidly become mono- and dinucleate by traction-mediated cell fission
and continue to grow vegetatively with a number of nuclei (1-2) per
cell, similar to that of wild-type cells.  The multinucleate
phenotype, combined with results indicating that constitutive
expression of activated Ras does not yield highly multinucleate cells
and data on Ras null mutants, suggest that Ras may need to cycle
between GTP- and GDP-bound states for proper cytokinesis.  After
starvation, the large null cells undergo rapid fission when they start
to move at the onset of aggregation, producing mononucleate cells that
form a normal aggregate.  Second, ddrasgap1 null cells also have
multiple developmental phenotypes that indicate an essential role of
DdRasGAP1 in controlling cell patterning.  Multicellular development
is normal through the mid-slug stage, after which morphological
differentiation is very abnormal and no culminant is formed: no stalk
cells and very few spores are detected.  LacZ reporter studies show
that by the mid-finger stage, much of the normal cell-type patterning
is lost, indicating that proper DdRasGAP1 function and possibly normal
Ras activity are necessary to maintain spatial organization and for
induction of prestalk -> stalk and prespore -> spore cell
differentiation.  The inability of ddrasgap1 null cells to initiate
terminal differentiation and form stalk cells is consistent with a
model in which Ras functions as a mediator of inhibitory signals in
cell-type differentiation at this stage.  Third, DdRasGAP1 and cAMP
dependent protein kinase (PKA) interact to control spatial
organization within the organism.  Overexpression of the PKA catalytic
subunit in ddrasgap1 cells yields terminal structures that are
multiply branched but lack spores.  This suggests that RasGAP and PKA
may mediate common pathways that regulate apical tip differentiation
and organizer function, which in turn control spatial organization
during multicellular development.  It also suggests that DdRasGAP1
either lies downstream from PKA in the prespore -> spore pathway or in
a parallel pathway that is also essential for spore differentiation.
Our results indicate that DdRasGAP1 plays an essential role in
controlling multiple, potentially novel pathways regulating growth and
differentiation in Dictyostelium and suggest a role for Ras in these
processes.

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[End CSM-News, volume 7, number 15]