- Email: [email protected]
Cyclic AMP dependent protein kinase and prestalk-cell gene expression in Dictyostelium
ELSEVIER
FEMS Microbiology
Letters I37 (1996) 275-278
Cyclic AMP dependent protein kinase and prestalk-cell gene expression in Dictyostelium Fabiana Horn, Julian D. Gross Department
qf Biochemistq.
Received 29 January
*
CJnil,ersiQ of Oxford. South Parks Roud. Oxford OXI 3QU UK 1996: revised 15 February
1996: accepted
15 February
1996
Abstract Cyclic AMP-dependent
protein kinase (PKA) is required for aggregation of starved amoebae during development of 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 (Nature 356 (1992) 17 l-172). 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.
Dictyostelium
discoideum
Keyword.r: Gene expression;
Protein kinase A; Dictyostelium
discoideum; Prestalk cell
1. Introduction During development, amoebae of Dictyostelium discoideum aggregate and form fruiting bodies made up of two major cell types, stalk cells and spores. Cyclic AMP serves as an extracellular chemotactic agent. Binding of CAMP to its cell surface receptors stimulates a number of second messenger pathways, among them activation of adenylyl cyclase; this in turn induces a transitory rise in intracellular cyclic AMP, followed by its release from the cells. Genetic and pharmacological evidence indicates that the
* Corresponding author. Tel.: +44 (1865) 275 238; Fax: f44 (1865) 275 259; e-mail: [email protected] 037%1097/96/$12.00 0 1996 Federation PI1 SO378- 1097(96)0007 l-7
of European
Microbiological
CAMP-dependent protein kinase (PKA) of Dictyostelium is required for aggregation and also plays an important role in prespore gene expression and in the formation of mature stalk cells and spores from prestalk and prespore cells, respectively [2,3]. Here we attempt to establish whether it also contributes to the ability of cells to express prestalk-cell-specific genes. Rapid developing (rde) mutants of Dictyostelium belonging to complementation group C have defects in the regulatory subunit of PKA, and hence exhibit unrestrained activity of the catalytic subunit [l]. During normal development on non-nutrient agar they form hemispherical mounds of cells containing mixtures of mature spores and stalk cells [4]. They are also ‘sporogenous’, that is, unlike the parental cells, Societies. All rights reserved
they form spores when incubated as submerged monolayers in the presence of cyclic AMP [S]. and they have been reported to produce stalk cells when starved in the absence of CAMP, though no quantitative information was given [6]. In order to define the point at which the stalk pathway is blocked in the parental cells, and so to identify the role of PKA in overcoming this block, we have examined cell-typespecific gene expression and cell differentiation in wild-type and rde cells under such conditions.
2. Materials
and methods
2.1. Struiru and culture conditions The D. discoideum rde- strain XPS.5. and strain HTY506, a rapidly developing rdeC mutant derived from it [4]. were used. Amoebae were grown in association with Klebsiellu aerogenes on SM agar plates at 22°C. harvested during exponential growth and freed from bacteria by differential centrifugations before being resuspended in the appropriate buffer. 2.2. In vitro d#erentiation Two millilitres of XPS5 or HTY506 cells resuspended in spore buffer (KC1 20.0 mM, NaCl 20.0 mM, MgSO, 1.O mM. CaCl? 1.O mM, dihydrostreptomycin sulfate 200 pug/ml. 10 mM Tris pH 7.5 [7]) were plated into 50 mm diameter NUNC culture dishes to give a final density of 5 X 10J cells pel
cm’. Where stated CAMP was added as a Kf salt at t,,. For RNA preparation, 15 ml of cell suspension was incubated in 140 mm diameter NUNC dishes. Cells were allowed to develop at 22°C. 2.3. RNA extraction
und Northern unalyis
For RNA preparation. cells were harvested from the NUNC dishes using a cell scraper and sedimented at 3000 rpm for 5 min. Total RNA was extracted by the guanidine thiocyanate/phenol-chloroform extraction method [8]. 5 pg aliquots of RNA were electrophoresed through a 1.5% agarose gel containing formaldehyde. The separated RNA was capillary blotted in 10 X SSC onto Hybond-N and fixed by baking at 80-90°C for 2 h. Samples were hybridised with radioactively labelled probes for the prestalk-specific marker ecmA [9] and presporespecific marker psA [lo].
3. Results and discussion We have used the submerged cell monolayer system [7,1 I], in which washed cells are incubated under buffer in tissue culture dishes, to compare the ability of an rdeC mutant (strain HTY506) and its parent (strain XP55) to express cell-type-specific genes and to form stalk cells. When plated at 2 X 10’ cells per cm’, the rdeC and rde+ amoebae aggregate at about the same time. The rdef amoebae form large streams and aggregates, while the rdeC amoebae form smaller aggregates (Fig. 1). In both cases
Fig. I. Aggregates of XPS5 (rde’) and HTY506 (rdeC) cells. XP5S and HTYS06 cells grown in association with K. trrro,qrrw, were freed from bacteria, plated in 35 mm diameter NUNC dishes at 2 X 10’ cell per cm’ in MES 5 mM. MgCI, 5 mM, KCI IO mM, pH 6.2 and incubated at 22°C. The aggregation process was observed under a NIKON SMZ-2B stereoscopic kroscope. The photographs shoti aggregating cells of (a) XPS5 and (b) HTY506 after I.5 h of development. XP55 bar = I mm; HTY506 bar = 250 Km.
F. Horn, J.D. Cross/
FEMS Microbiology
Table I Stalk cell and spore formation by XP55 (rde+ ) and HTY506 (rdeC) in the presence and absence of added CAMP
no CAMP 5 mMcAMP
XP55 (rde+ )
HTY506 (rdeC)
% stalk
9% spore
8 stalk
7c spore
0 0
0 0
31 0
7 15
Vegetative cells ofXP55 and HTY506 were plated in 50 mm diameter NUNC dishes at 5 X IO4 cell/cm’ in spore buffer pH 7.5. Where stated, CAMP was added at t,,. Cells were incubated at 22°C for 40 h and stained with calcofluor prior to scoring. The values are the mean of two experiments.
the aggregates evolve into loose clumps of cells displaying no further overt morphological development. When incubated at 5 X lo4 cells per cm’, the cells also aggregated and smaller clumps were formed. When the amoebae in the rdeC aggregates were observed at high power by phase contrast microscopy after 48 h incubation, it was found that many had transformed into stalk cells, and a smaller number into spores, whereas the cells of the parental strain remained amoeboid (Table 1). This finding confirms that when PKA is constitutively active all the processes required for formation of mature stalk
A.
0
XP55
Letten 137 (1996) 275-278
cells can be performed within submerged aggregates, even though CAMP is not provided externally. In order to test whether the rde+ amoebae were able to express prestalk-cell-specific genes under these conditions, we prepared RNA from wild-type and mutant cells incubated as monolayers in the presence and absence of CAMP, and hybridised it with probes for prespore and prestalk transcripts. As shown in Fig. 2, rde’ (XP55) cells failed to accumulate either prespore (psA) or prestalk (ecmA) transcripts when incubated in the absence of CAMP, whereas the rdeC cells (HTY506) accumulated significant levels of both transcripts. It is noteworthy that ecmA transcripts accumulated several hours be,fore psA transcripts whereas the converse is the case in normal development [13]. This accelerated prestalk gene expression was confirmed in a second experiment (not shown). When the monolayers were supplemented with millimolar CAMP the rde+ cells still failed to accumulate ecmA transcripts, probably due to CAMP inhibition of prestalk gene expression [ 141. They did, however, accumulate substantial levels of psA transcripts, consistent with the fact that such cells form prespore cells efficiently [15], although they were unable to mature into spores (Table I>. The rdeC cells again accumulated both ecmA and psA transcripts.
B. With CAMP
Without CAMP 6
12
16
20
277
0
24 h
8
12
16
20
24 h
- ecmA -
- psA -
HTY506
- ecmA -
- psA Fig. 2. Accumulation of ecmA and psA mRNA in XP55 (rde+) and HTY506 (rdeC). Cells (in spore buffer pH 7.5, at 5.4 X IO” cells per cm*) were allowed to differentiate in submerged cultures in the (A) absence and (B) presence of 5 mM CAMP at 22°C for the indicated times, Cells were then collected and total RNA was analysed as described in Section 2. Filters were probed for the prestalk-specific marker ecmA and prespore-specific marker psA. Probing for constitutively expressed I(37 mRNA 1121 confirmed that similar amounts of RNA were loaded in each lane (not shown).
p. Horn, J.D. Gloss / FEMS Microbiology
278
Our results indicate that when PKA activity is increased by blocking formation of the regulatory subunit, it permits prestalk gene expression under condition where the wild-type cells do not express prestalk genes. It is known that wild-type cells do express aggregative genes, so that the unregulated PKA activity must permit a transition from aggregative gene expression to prestalk gene expression. We cannot tell from our results whether PKA is directly involved in the ongoing transcription of prestalkcell-specific genes or is required for the expression or activation of a transcription factor required to initiate prestalk-cell-specific gene expression. However, the observation of reduced levels of ecmA transcripts in cells expressing a dominant-negative PKA regulatory subunit under the control of the ecmA promoter [16] supports the former possibility.
Lettrrs
[Al
I37 t IYY61 275%278
Abe. K. and Yanagisawa. developing
K. (1983)
A new class of rapid
mutants in Dicr~ostelium
tions for cyclic AMP
discoideum:
Implica-
metabolism and cell differentiation.
Dev. Biol. 95. 200-210. [51 Kay, R.R. t 1989) Evidence that elevated intracellular cyclic AMP triggers spore maturation in Dictymtelium. Development 105. 753-759.
[61 Abe,
K.. Saga. H., Okada.
Differentiation
K. t I98 I )
H. and Yanagisawa,
of Dic?\o,stdiwn
discoideum
mutant cells in a
shaken suspension culture and the effect of cyclic AMP.
J.
Cell Sci. 51. 131-132. [71 Kay. R.R. (1987) Cell differentiation in monolayers and the investigation of slime mold morphogens. In: Methods in Cell Biology (J.A. Spudich. Ed.) Vol. 28, pp. 433-448.
Academic
Press. London. [Xl Chomcrynaki. P. and Sacchi. N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162. I56- 159. [91 Jermyn, K.A.. Berks. M.. Kay, R.R. and Williams. J.G. (1987) Two distinct classes of prestalk-enriched mRNA sequences in
Dic~twstrlium
cli.sc~oideutm Development
100.
74.5-75s. [lOI Barklia, E. and Lodish. H.F. (1983) Regulation of !Iic.r~mteliunr discoideum mRNAs specific for prespore or pre-
Acknowledgements
stalk cells. Cell 32. 1139-l
This work was supported by a grant from the Wellcome Trust and Fabiana Horn acknowledges a studentship granted by CNPq (Brazilian Council for Scientific and Technological Development).
148.
[I 11 Town. CD.. Gross. J.D. and Kay, R.R. (1976) Cell differentiation without morphogenesis in Dictwstrliurn discviclrurt~. Nature 262. 717-719. [I21 Hopper, N.A.. Harwood. A.J.. Bouzid, S.. Veron, M. and Williams. J.G. (1993) Activation of the prespore and spore cell pathway of Dictwstrlium
differentiation
by CAMP-de-
pendent protein kinase and evidence for its upstream regulation by ammonia. EMBO
References
[ 131
Naylor. 0..
J. 12, 2459-2466.
Insall. R. and Kay. R.R. (1992)
Differentiation-
inducing-factor dechlorinase. a novel cytosolic dechlorinating [I]
Simon. M.N..
Pelegrini. 0.. Veron. M. and Kay. R.R. (1992)
enzyme from
Mutation of protein kinase A causes heterochronic develop-
208. 53 I-536.
ment of Dictwstrlium.
Nature 356. 17 I - 172.
Firtel. R.A.
t 1994) Expression of cAMP-dependent
differentiation
in
Dictyxtrlium.
protein
to induce spore cell
Proc. Natl. Acad. Sci. USA
91. 10561-10565. [31 Hopper.
N.A..
Combinatorial
cell differentiation by CAMP and DIF-I of Dictwstrlim
discoihrtn.
controlling
differentiation
in
control of
during development
Development
[I51 Kay, R.R. and Jermyn. K.A. (1983)
I IO, 9777984.
A possible morphogen
Dicfwstelinm.
Nature
303.
242-244. Sanders. G.M.,
Fosnaugh. K.L..
Williams.
[I61 Harwood, A.J., Hopper. N.A..
J.G. and Loomis. W.F. (1995) Protein kinase A is a positive
Veron,
regulator of spore coat gene transcription in I)ic.t\o.src,/ilrr,,.
tm~teliumis
Differentiation
cli,scoideuw~. Eur. J. Biochem.
[I-t] Berks. M. and Kay. R.R. (1990)
[2] Mann. S.K.O., Richardson, D.L.. Lee. S., Kimmel. A.R. and kinase in prespore cells is sufficient
Dictymtrliwtt
58.
I Xi-
188.
M. and William\.
Stmon. M.N.,
J.G. (1992)
Driscoll, D.M.,
Culmination
regulated hy the cAMP-dependent
nase. Cell 69. 615-621.
in Dir,-
protein kt-
FEMS Microbiology
Letters I37 (1996) 275-278
Cyclic AMP dependent protein kinase and prestalk-cell gene expression in Dictyostelium Fabiana Horn, Julian D. Gross Department
qf Biochemistq.
Received 29 January
*
CJnil,ersiQ of Oxford. South Parks Roud. Oxford OXI 3QU UK 1996: revised 15 February
1996: accepted
15 February
1996
Abstract Cyclic AMP-dependent
protein kinase (PKA) is required for aggregation of starved amoebae during development of 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 (Nature 356 (1992) 17 l-172). 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.
Dictyostelium
discoideum
Keyword.r: Gene expression;
Protein kinase A; Dictyostelium
discoideum; Prestalk cell
1. Introduction During development, amoebae of Dictyostelium discoideum aggregate and form fruiting bodies made up of two major cell types, stalk cells and spores. Cyclic AMP serves as an extracellular chemotactic agent. Binding of CAMP to its cell surface receptors stimulates a number of second messenger pathways, among them activation of adenylyl cyclase; this in turn induces a transitory rise in intracellular cyclic AMP, followed by its release from the cells. Genetic and pharmacological evidence indicates that the
* Corresponding author. Tel.: +44 (1865) 275 238; Fax: f44 (1865) 275 259; e-mail: [email protected] 037%1097/96/$12.00 0 1996 Federation PI1 SO378- 1097(96)0007 l-7
of European
Microbiological
CAMP-dependent protein kinase (PKA) of Dictyostelium is required for aggregation and also plays an important role in prespore gene expression and in the formation of mature stalk cells and spores from prestalk and prespore cells, respectively [2,3]. Here we attempt to establish whether it also contributes to the ability of cells to express prestalk-cell-specific genes. Rapid developing (rde) mutants of Dictyostelium belonging to complementation group C have defects in the regulatory subunit of PKA, and hence exhibit unrestrained activity of the catalytic subunit [l]. During normal development on non-nutrient agar they form hemispherical mounds of cells containing mixtures of mature spores and stalk cells [4]. They are also ‘sporogenous’, that is, unlike the parental cells, Societies. All rights reserved
they form spores when incubated as submerged monolayers in the presence of cyclic AMP [S]. and they have been reported to produce stalk cells when starved in the absence of CAMP, though no quantitative information was given [6]. In order to define the point at which the stalk pathway is blocked in the parental cells, and so to identify the role of PKA in overcoming this block, we have examined cell-typespecific gene expression and cell differentiation in wild-type and rde cells under such conditions.
2. Materials
and methods
2.1. Struiru and culture conditions The D. discoideum rde- strain XPS.5. and strain HTY506, a rapidly developing rdeC mutant derived from it [4]. were used. Amoebae were grown in association with Klebsiellu aerogenes on SM agar plates at 22°C. harvested during exponential growth and freed from bacteria by differential centrifugations before being resuspended in the appropriate buffer. 2.2. In vitro d#erentiation Two millilitres of XPS5 or HTY506 cells resuspended in spore buffer (KC1 20.0 mM, NaCl 20.0 mM, MgSO, 1.O mM. CaCl? 1.O mM, dihydrostreptomycin sulfate 200 pug/ml. 10 mM Tris pH 7.5 [7]) were plated into 50 mm diameter NUNC culture dishes to give a final density of 5 X 10J cells pel
cm’. Where stated CAMP was added as a Kf salt at t,,. For RNA preparation, 15 ml of cell suspension was incubated in 140 mm diameter NUNC dishes. Cells were allowed to develop at 22°C. 2.3. RNA extraction
und Northern unalyis
For RNA preparation. cells were harvested from the NUNC dishes using a cell scraper and sedimented at 3000 rpm for 5 min. Total RNA was extracted by the guanidine thiocyanate/phenol-chloroform extraction method [8]. 5 pg aliquots of RNA were electrophoresed through a 1.5% agarose gel containing formaldehyde. The separated RNA was capillary blotted in 10 X SSC onto Hybond-N and fixed by baking at 80-90°C for 2 h. Samples were hybridised with radioactively labelled probes for the prestalk-specific marker ecmA [9] and presporespecific marker psA [lo].
3. Results and discussion We have used the submerged cell monolayer system [7,1 I], in which washed cells are incubated under buffer in tissue culture dishes, to compare the ability of an rdeC mutant (strain HTY506) and its parent (strain XP55) to express cell-type-specific genes and to form stalk cells. When plated at 2 X 10’ cells per cm’, the rdeC and rde+ amoebae aggregate at about the same time. The rdef amoebae form large streams and aggregates, while the rdeC amoebae form smaller aggregates (Fig. 1). In both cases
Fig. I. Aggregates of XPS5 (rde’) and HTY506 (rdeC) cells. XP5S and HTYS06 cells grown in association with K. trrro,qrrw, were freed from bacteria, plated in 35 mm diameter NUNC dishes at 2 X 10’ cell per cm’ in MES 5 mM. MgCI, 5 mM, KCI IO mM, pH 6.2 and incubated at 22°C. The aggregation process was observed under a NIKON SMZ-2B stereoscopic kroscope. The photographs shoti aggregating cells of (a) XPS5 and (b) HTY506 after I.5 h of development. XP55 bar = I mm; HTY506 bar = 250 Km.
F. Horn, J.D. Cross/
FEMS Microbiology
Table I Stalk cell and spore formation by XP55 (rde+ ) and HTY506 (rdeC) in the presence and absence of added CAMP
no CAMP 5 mMcAMP
XP55 (rde+ )
HTY506 (rdeC)
% stalk
9% spore
8 stalk
7c spore
0 0
0 0
31 0
7 15
Vegetative cells ofXP55 and HTY506 were plated in 50 mm diameter NUNC dishes at 5 X IO4 cell/cm’ in spore buffer pH 7.5. Where stated, CAMP was added at t,,. Cells were incubated at 22°C for 40 h and stained with calcofluor prior to scoring. The values are the mean of two experiments.
the aggregates evolve into loose clumps of cells displaying no further overt morphological development. When incubated at 5 X lo4 cells per cm’, the cells also aggregated and smaller clumps were formed. When the amoebae in the rdeC aggregates were observed at high power by phase contrast microscopy after 48 h incubation, it was found that many had transformed into stalk cells, and a smaller number into spores, whereas the cells of the parental strain remained amoeboid (Table 1). This finding confirms that when PKA is constitutively active all the processes required for formation of mature stalk
A.
0
XP55
Letten 137 (1996) 275-278
cells can be performed within submerged aggregates, even though CAMP is not provided externally. In order to test whether the rde+ amoebae were able to express prestalk-cell-specific genes under these conditions, we prepared RNA from wild-type and mutant cells incubated as monolayers in the presence and absence of CAMP, and hybridised it with probes for prespore and prestalk transcripts. As shown in Fig. 2, rde’ (XP55) cells failed to accumulate either prespore (psA) or prestalk (ecmA) transcripts when incubated in the absence of CAMP, whereas the rdeC cells (HTY506) accumulated significant levels of both transcripts. It is noteworthy that ecmA transcripts accumulated several hours be,fore psA transcripts whereas the converse is the case in normal development [13]. This accelerated prestalk gene expression was confirmed in a second experiment (not shown). When the monolayers were supplemented with millimolar CAMP the rde+ cells still failed to accumulate ecmA transcripts, probably due to CAMP inhibition of prestalk gene expression [ 141. They did, however, accumulate substantial levels of psA transcripts, consistent with the fact that such cells form prespore cells efficiently [15], although they were unable to mature into spores (Table I>. The rdeC cells again accumulated both ecmA and psA transcripts.
B. With CAMP
Without CAMP 6
12
16
20
277
0
24 h
8
12
16
20
24 h
- ecmA -
- psA -
HTY506
- ecmA -
- psA Fig. 2. Accumulation of ecmA and psA mRNA in XP55 (rde+) and HTY506 (rdeC). Cells (in spore buffer pH 7.5, at 5.4 X IO” cells per cm*) were allowed to differentiate in submerged cultures in the (A) absence and (B) presence of 5 mM CAMP at 22°C for the indicated times, Cells were then collected and total RNA was analysed as described in Section 2. Filters were probed for the prestalk-specific marker ecmA and prespore-specific marker psA. Probing for constitutively expressed I(37 mRNA 1121 confirmed that similar amounts of RNA were loaded in each lane (not shown).
p. Horn, J.D. Gloss / FEMS Microbiology
278
Our results indicate that when PKA activity is increased by blocking formation of the regulatory subunit, it permits prestalk gene expression under condition where the wild-type cells do not express prestalk genes. It is known that wild-type cells do express aggregative genes, so that the unregulated PKA activity must permit a transition from aggregative gene expression to prestalk gene expression. We cannot tell from our results whether PKA is directly involved in the ongoing transcription of prestalkcell-specific genes or is required for the expression or activation of a transcription factor required to initiate prestalk-cell-specific gene expression. However, the observation of reduced levels of ecmA transcripts in cells expressing a dominant-negative PKA regulatory subunit under the control of the ecmA promoter [16] supports the former possibility.
Lettrrs
[Al
I37 t IYY61 275%278
Abe. K. and Yanagisawa. developing
K. (1983)
A new class of rapid
mutants in Dicr~ostelium
tions for cyclic AMP
discoideum:
Implica-
metabolism and cell differentiation.
Dev. Biol. 95. 200-210. [51 Kay, R.R. t 1989) Evidence that elevated intracellular cyclic AMP triggers spore maturation in Dictymtelium. Development 105. 753-759.
[61 Abe,
K.. Saga. H., Okada.
Differentiation
K. t I98 I )
H. and Yanagisawa,
of Dic?\o,stdiwn
discoideum
mutant cells in a
shaken suspension culture and the effect of cyclic AMP.
J.
Cell Sci. 51. 131-132. [71 Kay. R.R. (1987) Cell differentiation in monolayers and the investigation of slime mold morphogens. In: Methods in Cell Biology (J.A. Spudich. Ed.) Vol. 28, pp. 433-448.
Academic
Press. London. [Xl Chomcrynaki. P. and Sacchi. N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162. I56- 159. [91 Jermyn, K.A.. Berks. M.. Kay, R.R. and Williams. J.G. (1987) Two distinct classes of prestalk-enriched mRNA sequences in
Dic~twstrlium
cli.sc~oideutm Development
100.
74.5-75s. [lOI Barklia, E. and Lodish. H.F. (1983) Regulation of !Iic.r~mteliunr discoideum mRNAs specific for prespore or pre-
Acknowledgements
stalk cells. Cell 32. 1139-l
This work was supported by a grant from the Wellcome Trust and Fabiana Horn acknowledges a studentship granted by CNPq (Brazilian Council for Scientific and Technological Development).
148.
[I 11 Town. CD.. Gross. J.D. and Kay, R.R. (1976) Cell differentiation without morphogenesis in Dictwstrliurn discviclrurt~. Nature 262. 717-719. [I21 Hopper, N.A.. Harwood. A.J.. Bouzid, S.. Veron, M. and Williams. J.G. (1993) Activation of the prespore and spore cell pathway of Dictwstrlium
differentiation
by CAMP-de-
pendent protein kinase and evidence for its upstream regulation by ammonia. EMBO
References
[ 131
Naylor. 0..
J. 12, 2459-2466.
Insall. R. and Kay. R.R. (1992)
Differentiation-
inducing-factor dechlorinase. a novel cytosolic dechlorinating [I]
Simon. M.N..
Pelegrini. 0.. Veron. M. and Kay. R.R. (1992)
enzyme from
Mutation of protein kinase A causes heterochronic develop-
208. 53 I-536.
ment of Dictwstrlium.
Nature 356. 17 I - 172.
Firtel. R.A.
t 1994) Expression of cAMP-dependent
differentiation
in
Dictyxtrlium.
protein
to induce spore cell
Proc. Natl. Acad. Sci. USA
91. 10561-10565. [31 Hopper.
N.A..
Combinatorial
cell differentiation by CAMP and DIF-I of Dictwstrlim
discoihrtn.
controlling
differentiation
in
control of
during development
Development
[I51 Kay, R.R. and Jermyn. K.A. (1983)
I IO, 9777984.
A possible morphogen
Dicfwstelinm.
Nature
303.
242-244. Sanders. G.M.,
Fosnaugh. K.L..
Williams.
[I61 Harwood, A.J., Hopper. N.A..
J.G. and Loomis. W.F. (1995) Protein kinase A is a positive
Veron,
regulator of spore coat gene transcription in I)ic.t\o.src,/ilrr,,.
tm~teliumis
Differentiation
cli,scoideuw~. Eur. J. Biochem.
[I-t] Berks. M. and Kay. R.R. (1990)
[2] Mann. S.K.O., Richardson, D.L.. Lee. S., Kimmel. A.R. and kinase in prespore cells is sufficient
Dictymtrliwtt
58.
I Xi-
188.
M. and William\.
Stmon. M.N.,
J.G. (1992)
Driscoll, D.M.,
Culmination
regulated hy the cAMP-dependent
nase. Cell 69. 615-621.
in Dir,-
protein kt-