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First published online 31 January 2007
doi: 10.1242/dev.02775
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Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, Angus DD1 5EH, UK.
* Author for correspondence (e-mail: p.schaap{at}dundee.ac.uk)
Accepted 5 December 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Pattern formation, Cell-type specification, Sporulation, Encystation, Adenylyl cyclase G, Dictyostelium discoideum
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Infections with Acanthamoeba
castellani, which cause keratitis and amoebic encephalitis, are difficult
to treat because the amoebae differentiate into highly resistant cysts inside
host tissues (Lloyd et al.,
2001; Marciano-Cabral and
Cabral, 2003; McClellan et
al., 2002). Little is known of the signalling pathways that
control encystation, mainly due to lack of genetic tools to investigate this
process.
Social amoebae respond to nutrient stress by either encysting individually
or by aggregating to form fruiting structures, where most of the cells
differentiate into spores. A small proportion of cells altruistically build a
stalk to support the spore mass and to aid in their dispersal. In particular,
the species Dictyostelium discoideum has excellent genetic
tractability, and the pathways that control sporulation have been extensively
studied. Here, sporulation involves a first phase, prespore differentiation
that occurs shortly after aggregation. In this stage the cells synthesize
spore-coat components in prespore vesicles, but remain otherwise amoeboid.
Prespore differentiation is triggered by extracellular cAMP acting on cAMP
receptors (cARs), and intracellular cAMP acting on protein kinase (PKA)
(Schaap and Van Driel, 1985;
Hopper et al., 1993). The
second phase, spore maturation, occurs after the stalk is formed and this
process is triggered solely by a high level of PKA activity
(Mann et al., 1994). Spore
maturation involves relatively minor changes in gene expression, but is
accompanied by major physiological changes: prespore vesicles fuse with the
plasma membrane, laying down the first layers of the spore coat and releasing
precursors for synthesis of the outer layers
(West and Erdos, 1990).
PKA activation during spore maturation requires the activity of the
adenylyl cyclase ACB, encoded by AcrA, which is maximally expressed
at the fruiting body stage (Kim et al.,
1998; Meima and Schaap,
1999; Soderbom et al.,
1999). In addition, the process requires inactivation of the
intracellular cAMP phosphodiesterase, RegA. This unusual enzyme harbours a
response regulator domain which is the target of a phosphorelay system that is
regulated by sensor histidine kinases/phosphatases
(Shaulsky et al., 1996;
Shaulsky et al., 1998;
Thomason et al., 1998;
Thomason et al., 1999). A
peptide released by stalk cells, SDF-2, activates the sensor histidine
phosphatase DhkA, causing dephosphorylation and hence inactivation of RegA.
This in turn causes cAMP accumulation and the activation of PKA
(Anjard and Loomis, 2005;
Wang et al., 1999). PKA
remains important in the spore stage, where it controls spore dormancy. The
ambient high osmolality in the spore head keeps the spores dormant, and this
effect is mediated by the adenylyl cyclase ACG, which harbours an
intramolecular osmosensor (Saran and
Schaap, 2004; Van Es et al.,
1996; Virdy et al.,
1999).
The requirements of ACB and ACG for PKA activation in spore maturation and
dormancy are well documented. However, it is not clear which enzyme produces
the extracellular cAMP that triggers prespore differentiation. The third
Dictyostelium adenylyl cyclase, ACA, is mainly active during
aggregation and disappears from the prespore region once slugs start to form
(Pitt et al., 1992;
Verkerke-van Wijk et al.,
2001). Null mutants in ACB/AcrA show normal
prespore gene expression (Soderbom et al.,
1999), and ACG mRNA was only detectable in spores
(Pitt et al., 1992). However,
biochemical analysis of adenylyl cyclase activities in aca- slugs
demonstrated the presence of an adenylyl cyclase activity, which as with ACG,
preferred Mn2+-ATP over Mg2+-ATP as a substrate. The
reverse is true for ACB, which suggests that ACG could be expressed in slugs
(Meima and Schaap, 1999).
In this work we analysed the pattern of ACG transcription and translation more closely by studies with ACG promoter-reporter gene fusions and an ACG-specific antibody. Our data indicate that ACG is transcribed at low levels throughout development, whereas ACG protein is markedly upregulated after aggregation in the prespore regions of slugs. Analysis of single and double null mutants in ACG and ACB indicates that ACG is essential for prespore differentiation, but that its function is partially redundant with ACB. This work complements parallel studies in which we show that ACG is deeply conserved in amoebazoan evolution and regulates encystation and excystation in analogy to its roles in spore formation and germination.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To induce competence for prespore gene induction, cells were starved on PB agar for 16 hours at 6oC and 2 hours at 22oC until aggregation territories had formed. Cells were then resuspended to 2x106 cells/mL in PB and shaken at 150 rpm and 22oC in the presence and absence of cAMP.
Gene constructs and transformation
Fusion constructs of the ACG promoter were made with the lacZ
(gal) reporter gene and with a modified lacZ, called ile-gal. In
ile-gal, lacZ is modified by N-terminal addition of the ubiquitin
gene and replacement of the lacZ start codon with an isoleucine
codon. The ubiquitin moiety is cleaved off during translation, leaving
ß-galactosidase with an exposed isoleucine, which decreases protein
stability to a half-life of 30 minutes
(Detterbeck et al., 1994). For
both constructs, 2855 bp of ACG DNA sequence, comprising 2810 bp of the
complete 5' intergenic region and 45 bp of coding sequence, were
amplified from vector pGACG (Pitt et al.,
1992) using primers ACGpr5' and ACGpr3'
(Table 1), which harbour
XbaI and BglII sites, respectively. After digestion with
XbaI and BglII, the amplified product was cloned into
XbaI/BglII digested pDdGal-17
(Harwood and Drury, 1990) to
create ACG::gal, and used to replace the XbaI/BglII psA
promoter fragment from vector psA-ile-gal
(Detterbeck et al., 1994) to
generate ACG::ile-gal. The vectors were introduced into AX3 cells and
acrA- mutants by electroporation, and transformants were selected for
growth at 100 µg/mL G418 for ACG::gal and at 200 µg/mL G418 for the
ACG::ile-gal constructs.
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) were made by amplification of the 819 bp AcrA
5' intergenic region from AX2 genomic DNA with primers AcrApr5'
and AcrApr3', containing XbaI and BglII restriction
sites (Table 1). The amplifed
product was inserted into both the ile-gal and ala-gal vector as described
above to create AcrA-ile-gal and AcrA-ala-gal. Both vectors were introduced
into AX2 and acg- cells. Transformants were selected for growth at
100 µg/mL G418.
To prepare an ACG gene disruption construct, two DNA fragments of the
acgA gene comprising nucleotides 29-922 and 1761-2184 were amplified
by PCR from vector pGACG (Pitt et al.,
1992), using oligonucleotides AcgKO1-4
(Table 1) that add a
5'-BamHI and 3'-KpnI site to the first fragment
and a 5'-XbaI and 3'-BamHI site to the second fragment.
These fragments were cloned sequentially into BamHI/KpnI
digested and XbaI/BamHI digested pBsr
Bam
(Sutoh, 1993). The construct
was linearized with BamHI, which yielded the pBsrBam plasmid
flanked by 894 bp and 423 bp of 5' and 3' AcgA sequence,
respectively, and introduced into wild-type AX2 cells. Transformed cells were
selected for growth at 5 µg/mL blasticidin, and selected clones were
screened for homologous recombination by two separate PCR reactions and
analysis of Southern blots of genomic digests.
Histochemical and spectrophotometric ß-galactosidase assays
For visualization of ß-galactosidase activity in developing
structures, cells were distributed at 107 cells/cm2 on
nitrocellulose filters supported by PB agar and incubated at 22°C.
Structures were fixed in 0.25% glutaraldehyde, containing 2% Tween-20 and
stained with X-Gal as described previously
(Dingermann et al., 1989).
For spectrophotometric measurement of ß-galactosidase activity, cells
were lysed by freeze-thawing and 100 µL aliquots of lysate were incubated
at 22°C in microtitre plate wells with 30 µL of 2.5xZ-buffer and
20 µL of 40 mM chlorophenolred-ß-D-galactopyranoside
(Schaap et al., 1993). The
OD574 was measured at regular time intervals using a microtitre
plate reader; ß-galactosidase activity in OD574/minute was
calculated from the time intervals where reaction product accumulated linearly
and was standardized on the protein content of the samples. The activity
observed in untransformed cells was subtracted as the assay blank.
Immunological techniques
For immunoblotting, samples of 2x107 cells were pelleted
and boiled in 50 µL SDS sample buffer. Samples of 50 µg of total protein
were size-fractionated on 8% SDS-PAA gels and transferred to nitrocellulose
membranes. Membranes were incubated overnight at 4°C with a 1:2000
dilution of an 
ACG peptide antibody
(Saran and Schaap, 2004),
washed and incubated with 1:2000 diluted horseradish peroxidase-coupled
goat-anti-rabbit antibody (Promega, USA). Detection was performed with the
Supersignal chemoluminescence kit (Pierce, USA) according to the
manufacturer's instructions.
For immunocytochemistry, slugs were harvested in 20 mM EDTA in PB and
dissociated into single cells by passing through a 23-gauge needle. Cells were
placed as 10 µL aliquots of 107 cells/mL on eight-well multitest
slides, overlayed with agarose (Fukui et
al., 1986) and fixed for 10 minutes in ice-cold methanol. Slides
were incubated overnight with 1:500 diluted ACG antibody, and with
1:200 diluted FITC-conjugated goat-anti-rabbit IgG (GARFITC) for 1 hour.
Subsequently cells were incubated for 1 hour with a 1:500 diluted mouse
monoclonal antibody 83.5 (Zhang et al.,
1999) and for 1 hour with 1:500 diluted Texas Red-conjugated goat
anti-mouse IgG. Spores were harvested from fruiting bodies and stained with
ACG antibody and GARFITC.
For whole-mount immunostaining, intact structures were gently floated from
an inverted slice of supporting agar to 10 µL PB deposited in the wells of
polylysine-coated eight-well multitest slides. The fluid was aspirated and the
structures were fixed in methanol and incubated with ACG antibody and
GARFITC as described above. Preparations were photographed using a Leica TCS
SP2 confocal laser scanning microscope.
To measure the proportion of prespore cells, fully migrating slugs were
dissociated into single cells by repeated aspiration in 1% (w/v) cellulase in
2 mM EDTA, pH 6.5. Cells were then fixed in methanol and incubated for 16
hours at 4°C with 1:50 diluted spore-antiserum
(Takeuchi, 1963) and for 1
hour with 1:200 diluted GARFITC. The samples were counterstained with 1
µg/mL of 4,6-diaminidino-2-phenylindole (DAPI). Cells were photographed
using a Leica DM LB2 fluorescence microscope, and total cells (DAPI-stained)
and prespore cells (cells with >3 FITC-stained vesicles) were counted.
RNA detection by in-situ hybridization and RT-PCR
In-situ hybridization
Cells were incubated at 106 cells/cm2 on dialysis
membrane, supported by PB agar, until the desired developmental stages had
been reached. In-situ hybridization with 200 ng/mL of digoxigenin
(DIG)-labelled AcrA RNA was carried out as previously described
(Escalante and Loomis, 1995).
An antisense AcrA probe was used as a control. To prepare the probes,
a 520 bp AcrA fragment was amplified from genomic DNA using primers
AcrAcat5' and AcrAcat3' (Table
1) and cloned into EcoRI/BamHI digested pBluescript KS+. The
AcrA fragment was subsequently amplified by PCR using the universal
M13-20 and `Reverse' primers. The purified PCR product served as template for
synthesis of sense and antisense DIG-labelled AcrA RNA probes using
the SP6 and T7 RNA polymerases and reagents from a DIG RNA labelling kit
(Roche, UK).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This does
not preclude expression at an earlier stage, because 2-3-fold lower levels
would go undetected in northern blots. To obtain more information on the
spatiotemporal pattern of ACG transcription, we fused 2.8 kb of 5'
flanking sequence of the ACG gene to the ß-galactosidase (gal) reporter
gene. The parent strain AX3 was transformed with the ACG::gal
construct and developing structures were stained with X-Gal for
ß-galactosidase activity. Surprisingly, ß-galactosidase activity was
already present in aggregating cells and newly formed slugs, although activity
was most pronounced in the spore head of fruiting bodies
(Fig. 1A-C). Because the
ß-galactosidase protein is stable, it will progressively accumulate in
cells, even if gene transcription is low. To investigate whether this caused
the discrepancy between the ACG::gal and earlier mRNA data, we made a
second gene fusion of the ACG promoter with ile-gal that encodes a labile
ß-galactosidase protein (Detterbeck et
al., 1994). In cells transformed with this construct,
ß-galactosidase activity was barely detectable in slugs
(Fig. 1D), but did become
visible in the prespore region of mid-culminants
(Fig. 1E). This indicates that
ACG promoter activity must be low during early development and only increases
significantly at the onset of fruiting body formation.
ACG protein levels during development
We next measured the developmental regulation of ACG protein expression by
immunoblotting using an ACG peptide antibody that was raised and tested for
specificity previously (Saran and Schaap,
2004). Fig. 2A
shows that in wild-type cells, ACG protein levels rapidly increased during tip
formation to reach a plateau in migrating slugs. Fruiting body formation was
accompanied by a further modest increase in ACG protein levels. This
expression pattern is more consistent with the expression of prespore genes
than that of spore genes. To test this further we measured ACG expression in
an ACB/acrA null mutant (Kim et
al., 1998; Soderbom et al.,
1999). ACB is essential for the expression of spore genes, but not
for the differentiation of prespore cells. However, also in the acrA-
mutant, ACG protein accumulated rapidly during tip formation, with only a
minor increase in mature fruiting bodies
(Fig. 2B).
The profiles of ACG protein accumulation measured here, and ACG
mRNA measured earlier (Pitt et al.,
1992) are quite different, with mRNA only being detected in the
spore stage. We used the more sensitive reporter gene assay to determine the
developmental profile of ACG promoter activity in wild-type and
acrA- cells, both transformed with the ACG::ile-gal constructs.
Fig. 2C shows that consistent
with the earlier data, ACG promoter activity, shows a dramatic
increase during fruiting body formation. However, there is low but detectable
activity during the entire course of development. This explains why stable
ß-galactosidase protein could accumulate in early development
(Fig. 1A,B). Both ACG
promoter activity and ACG protein synthesis were normal in acrA-
cells. This indicates that unlike other spore genes
(Soderbom et al., 1999), the
expression of ACG is not dependent on ACB activity.
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Localization of ACG in cells and tissues
To gain insight into the role of ACG in slugs, we first visualized the
pattern of ACG protein expression. Fig.
3A shows that in newly formed slugs ACG protein was exclusively
localized at the posterior prespore region. In mid-culminants, ACG protein was
highly expressed throughout the prespore region, but was absent from the
stalk, prestalk and lower cup regions (Fig.
3B). In spores, ACG was localized at the cell periphery as would
be expected for a transmembrane osmosensor
(Fig. 3C). However, in slugs
ACG staining was distributed in a punctuated fashion over the cells,
reminiscent of the distribution of prespore vesicles. To test this we double
stained slug cells with ACG antibody
(Fig. 3D) and with an
SP85 antibody (Zhang et al.,
1999) (Fig. 3E).
SP85 is a spore-coat protein that is associated with prespore vesicles
(Zhang et al., 1998). The
superimposed image (Fig. 3F)
shows that ACG and SP85 are colocalized in the same cellular compartments,
which are most likely the prespore vesicles.
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;
Wang et al., 1988) and/or
intracellular cAMP for PKA activation, that is required for expression of a
subset of prespore genes (Hopper et al.,
1993). Null mutants in ACG were originally described to form
fruiting bodies normally, but structures were not studied in great detail
(Pitt et al., 1992). We
compared prespore and spore differentiation in null mutants for ACG, ACB/AcrA
and in a mutant that has neither activity. This mutant was made by expressing
ACGcat, a dominant negative inhibitor of ACG
(Saran and Schaap, 2004) in
acrA- cells under the constitutive actin15 promoter. A new
acg- mutant was created because fruiting body formation in the
original mutant has deteriorated over time.
To estimate effects of the mutations on prespore differentiation, we
measured both the proportion of prespore cells in dissociated slugs and the
expression of the prespore gene CotB
(Fosnaugh and Loomis, 1993;
Gomer et al., 1986) during
normal development to fruiting bodies. Fig.
4A shows that the percentage of prespore to total cells was
reduced from 64% to 50% in acrA- cells and to 40% in acg-
cells. The most severe reduction to about one-third of wild-type prespore
proportions is observed in the acrA-/ACGcat cells.
The developmental expression of the prespore gene CotB showed a
similar pattern (Fig. 4B). In
acrA- cells, cotB expression was slightly reduced, in
acg- cells reduction was more severe and in
acrA-/ACGcat cells CotB mRNA was almost
gone.
All three mutant cell lines still formed fruiting bodies. As previously
reported (Soderbom et al.,
1999), the spore heads of mature acrA- fruiting bodies
contained large numbers of amoeboid cells and only a few spores
(Fig. 4C). In contrast, most
cells in the acg- spore heads had matured into spore cells. However,
in the acrA-/ACGcat spore heads only a few spores and
several empty spore cases were visible. The remaining spores were extremely
fragile and often ruptured while being carried over on a slide glass for
observation.
These combined data show that loss of ACG is most deleterious for prespore differentiation, whereas loss of ACB has the strongest effect on spore maturation. However, the two enzymes show considerable functional redundancy, and the most severe phenotypes on both prespore and spore differentiation are evident when they are both lost.
cAMP induction of prespore gene expression in adenylyl cyclase mutants
The induction of most prespore genes, such as CotB, requires both
extracellular cAMP acting on cARs and intracellular cAMP acting on PKA
(Hopper et al., 1995;
Schaap and Van Driel, 1985).
However, the prespore gene PsA is less sensitive to ablation of PKA
function (Hopper et al., 1993).
To examine whether ACG and/or ACB mediate both the intracellular and
extracellular functions of cAMP, we measured to what extent CotB and
PsA gene expression were restored by extracellular cAMP in the
adenylyl cyclase null mutants. Fig.
5 shows that PsA gene expression is almost fully restored
by extracellular cAMP in both the acrA-, acg- and
acrA-/ACGcat mutants. However, CotB
induction was reduced in the acrA- and acg- mutant and
almost absent in the acrA-/ACGcat mutant. These
results indicate that ACG and ACB have overlapping roles in both cAR and PKA
activation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Saran and Schaap, 2004;
Van Es et al., 1996). Our
present data confirm that ACG gene expression is strongly upregulated
in maturing fruiting bodies; however, there is also significant transcription
throughout development. Remarkably, ACG protein is upregulated 12 hours before
fruiting bodies are formed in the absence of a corresponding increase in
transcription. ACG protein first appears in tipped mounds, to accumulate later
in the prespore region of slugs, where it colocalizes with the prespore
vesicles. At this location the ACG sensor domain would face the lumen of the
vesicle and its catalytic domain would face the cytosol. When prespore
vesicles fuse with the plasma membrane in the course of spore maturation, the
ACG sensor domain becomes exposed to the exterior of the cell.
Significant ACG-like activity (1.7 pmol cAMP/min.107cells) could
previously be detected in slug lysates
(Meima and Schaap, 1999)
(M.M., unpublished data), but no osmostimulation of ACG was detectable in
intact slug cells. This indicates that the vesicular localization of ACG does
not interfere with its enzyme activity, because the catalytic domain would
still be exposed to the substrate Mg2+-ATP in the cytosol. However,
osmostimulation may either not be possible, or, dependent on the ambient
osmolality in the prespore vesicles, the enzyme may always be in the
stimulated state. Most of the cAMP that is produced by any of the three
Dictyostelium adenylyl cyclases is rapidly secreted, suggesting a
general non-adenylyl cyclase-dependent mechanism for cAMP secretion
(Meima and Schaap, 1999;
Pitt et al., 1992). This
implies that as long as cAMP is produced in the cytosol, it can act both as an
intracellular and extracellular signal by virtue of its constitutive
secretion.
Early work showed that extracellular cAMP is both necessary and sufficient
for prespore gene induction: micromolar cAMP acting on surface cAMP receptors
triggers prespore differentiation (Schaap
and Van Driel, 1985), whereas depletion of extracellular cAMP in
slugs causes dedifferentiation of prespore cells
(Wang et al., 1988). However,
it was less clear how micromolar cAMP concentrations are being produced in
slug posteriors. The aggregation-specific adenylyl cyclase ACA is
downregulated in slugs and remains only expressed in the tip
(Verkerke-van Wijk et al.,
2001). AcrA null mutants are defective in spore
maturation, but not in prespore differentiation
(Soderbom et al., 1999). We
show here that prespore differentiation is significantly reduced in
acg- cells and has almost disappeared from mutants where both ACG and
ACB function is abrogated. Such mutants also do not form any mature spores.
These data indicate that ACG and ACB play combinatorial roles in prespore and
spore differentiation with ACG predominantly responsible for the former and
ACB for the latter response.
Surprisingly AcrA/ACB is specifically expressed in prestalk cells, which suggests that its effects on spore maturation may be indirect. In the absence of ACG, AcrA/ACB becomes expressed throughout the prespore region, which adequately explains why prespore differentiation is only partially lost in acg- cells. The low residual level of prespore gene expression that is still present in slugs where both ACG and ACB function are abrogated, could be due to the remaining enzyme ACA.
Expression of the majority of prespore genes not only requires
extracellular cAMP acting on cAMP receptors, but also intracellular cAMP
acting on PKA (Hopper et al.,
1993). We show that ACG produces cAMP for both functions
(Fig. 5), and it was previously
shown to produce cAMP for PKA activation in the spore stage. Here ACG acts as
a sensor for the high level of osmolytes in the spore head, which serves to
keep the spores dormant (Saran and Schaap,
2004; Van Es et al.,
1996; Virdy et al.,
1999). Recent work in our laboratory indicates that the ACG gene
has been conserved throughout the Dictyostelid phylogeny (A. V. Ritchie and
P.S., unpublished). In addition to spore formation in fruiting bodies, many
Dictyostelid species can encyst as single cells, which represents the survival
strategy of their ancestors, the solitary amoebae
(Raper, 1984). The encystation
process is triggered by high osmolality and requires activation of PKA (A. V.
Ritchie and P.S., unpublished). It therefore appears that the role of ACG in
prespore differentiation and spore dormancy is derived from a deeply conserved
role in encystation.
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ACKNOWLEDGMENTS |
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