scholarly journals Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor.

1987 ◽  
Vol 7 (1) ◽  
pp. 458-469 ◽  
Author(s):  
S K Mann ◽  
R A Firtel
Keyword(s):  
Cyclic Amp ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Early Gene ◽  
Developmental Cycle ◽  
Intracellular Camp ◽  
Flanking Sequence ◽  
Coding Region ◽  
Camp Receptor

We examined two sets of genes expressed early in the developmental cycle of Dictyostelium discoideum that appear to be regulated by cyclic AMP (cAMP). The transcripts of both sets of genes were not detectable in vegetative cells. During normal development on filter pads, RNA complementary to these genes could be detected at about 2 h, peaked around 6 to 8 h, and decreased gradually thereafter. Expression of these genes upon starvation in shaking culture was stimulated by pulsing the cells with nanomolar levels of cAMP, a condition that mimics the in vivo pulsing during normal aggregation. Expression was inhibited by caffeine or by continuous levels of cAMP, a condition found later in development when in vivo expression of these genes decreased. The inhibition of caffeine could be overcome by pulsing cells with cAMP. These results suggest that the expression is mediated via the cell surface cAMP receptor, but does not require a rise in intracellular cAMP. mRNA from a gene of the second class was induced upon starvation, peaked by 2.5 h of development, and then declined. In contrast to the other genes, its expression was maintained by continuous levels of cAMP and repressed by cAMP pulses. These and other results on a number of classes of developmentally regulated genes indicates that changing levels of cAMP, acting via the cell surface cAMP receptor, are involved in controlling these groups of genes. We also examined the structure and partial sequence of the cAMP pulse-induced genes. The two tandemly duplicated M3 genes were almost continuously homologous over the sequenced portion of the protein-coding region except for a region near the N-terminal end. The two M3 genes had regions of homology in the 5' flanking sequence and showed slight homology to the same regions in gene D2, another cAMP pulse-induced gene. D2 showed extremely significant homology over its entire sequenced length to an acetylcholinesterase. The results presented here and by others suggest that expression of many early genes in D. discoideum is regulated via the cell surface cAMP receptor. We expect that many of these genes may play essential roles in early Dictyostelium development and could code for elements of the cAMP signal transduction pathway involved in aggregation.

Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor

1987 ◽  
Vol 7 (1) ◽  
pp. 458-469
Author(s):  
S K Mann ◽  
R A Firtel
Keyword(s):  
Cyclic Amp ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Early Gene ◽  
Developmental Cycle ◽  
Intracellular Camp ◽  
Flanking Sequence ◽  
Coding Region ◽  
Camp Receptor

We examined two sets of genes expressed early in the developmental cycle of Dictyostelium discoideum that appear to be regulated by cyclic AMP (cAMP). The transcripts of both sets of genes were not detectable in vegetative cells. During normal development on filter pads, RNA complementary to these genes could be detected at about 2 h, peaked around 6 to 8 h, and decreased gradually thereafter. Expression of these genes upon starvation in shaking culture was stimulated by pulsing the cells with nanomolar levels of cAMP, a condition that mimics the in vivo pulsing during normal aggregation. Expression was inhibited by caffeine or by continuous levels of cAMP, a condition found later in development when in vivo expression of these genes decreased. The inhibition of caffeine could be overcome by pulsing cells with cAMP. These results suggest that the expression is mediated via the cell surface cAMP receptor, but does not require a rise in intracellular cAMP. mRNA from a gene of the second class was induced upon starvation, peaked by 2.5 h of development, and then declined. In contrast to the other genes, its expression was maintained by continuous levels of cAMP and repressed by cAMP pulses. These and other results on a number of classes of developmentally regulated genes indicates that changing levels of cAMP, acting via the cell surface cAMP receptor, are involved in controlling these groups of genes. We also examined the structure and partial sequence of the cAMP pulse-induced genes. The two tandemly duplicated M3 genes were almost continuously homologous over the sequenced portion of the protein-coding region except for a region near the N-terminal end. The two M3 genes had regions of homology in the 5' flanking sequence and showed slight homology to the same regions in gene D2, another cAMP pulse-induced gene. D2 showed extremely significant homology over its entire sequenced length to an acetylcholinesterase. The results presented here and by others suggest that expression of many early genes in D. discoideum is regulated via the cell surface cAMP receptor. We expect that many of these genes may play essential roles in early Dictyostelium development and could code for elements of the cAMP signal transduction pathway involved in aggregation.

Pharmacological characterization of cyclic AMP receptors mediating gene regulation in Dictyostelium discoideum

1986 ◽  
Vol 6 (7) ◽  
pp. 2402-2408
Author(s):  
B Haribabu ◽  
R P Dottin
Keyword(s):  
Gene Expression ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Cell Surface Receptor ◽  
Intracellular Camp ◽  
Response Curves ◽  
Pharmacological Characterization ◽  
Surface Receptor ◽  
Extracellular Molecules

Extracellular molecules regulate gene expression in eucaryotes. Exogenous cyclic AMP (cAMP) affects the expression of a large number of developmentally regulated genes in Dictyostelium discoideum. Here, we determine the specificity of the receptor(s) which mediates gene expression by using analogs of cAMP. The order of potency with which these analogs affect the expression of specific genes is consistent with the specificity of their binding to a cell surface receptor and is distinct from their affinity for intracellular cAMP-dependent protein kinase. Dose-response curves with cAMP and adenosine 3',5'-monophosphorothioate, a nonhydrolyzable analog, revealed that the requirement for high concentrations of exogenous cAMP for regulating gene expression is due to the rapid degradation of cAMP by phosphodiesterase. The addition of low concentrations of cAMP (100 nM) or analogs in pulses also regulates gene expression. Both the genes that are positively regulated by exogenous cAMP and the discoidin gene, which is negatively regulated, respond to cAMP analogs to the same degree. Genes expressed in prespore or prestalk cells are also similarly regulated. These data suggest that the effects are mediated through the same receptor. The specificity of this receptor is indistinguishable from that of the well-characterized cell surface cAMP receptor.

scholarly journals A pharmacologically distinct cyclic AMP receptor is responsible for the regulation of gp80 expression in Dictyostelium discoideum.

1990 ◽  
Vol 10 (7) ◽  
pp. 3297-3306 ◽  
Author(s):  
P C Ma ◽  
C H Siu
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Dictyostelium Discoideum ◽  
Surface Glycoprotein ◽  
Intracellular Camp ◽  
Cell Surface Glycoprotein ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation ◽  
Camp Receptor

The EDTA-resistant cell-cell adhesion expressed at the aggregation stage of Dictyostelium discoideum is mediated by a cell surface glycoprotein of Mr 80,000 (gp80). The expression of gp80 is developmentally regulated by cyclic AMP (cAMP). In vitro nuclear run-on experiments show that transcription of the gp80 gene is initiated soon after the onset of development. The basal level of gp80 transcription is significantly augmented by exogenous cAMP pulses. Interestingly, in analog studies, 2'-deoxy-cAMP, 8-bromo-cAMP, and N6-monobutyryl-cAMP are all capable of inducing a rapid accumulation of gp80 mRNA, suggesting the presence of a unique cAMP receptor that responds equally well to these analogs. To determine whether intracellular cAMP plays a role in the regulation of gp80 expression, caffeine was used to block cAMP-induced receptor-mediated adenylate cyclase activation. Expression of gp80 mRNA was blocked in caffeine-treated cells but could be substantially restored by treatment with exogenous cAMP pulses, suggesting that adenylate cyclase activation is not required. gp80 expression was also examined in the signal transduction mutants synag 7 and frigid A. In both mutants, gp80 was expressed at the basal level. Pulses of cAMP as well as 2'-deoxy-cAMP and N6-monobutyryl-cAMP were capable of restoring the normal level of gp80 expression in synag 7 cells. These results, taken together, indicate bimodal regulation of gp80 expression during development and the involvement of a novel cAMP receptor in the transmembrane signalling pathway that regulates gp80 gene expression.

scholarly journals Pharmacological characterization of cyclic AMP receptors mediating gene regulation in Dictyostelium discoideum.

1986 ◽  
Vol 6 (7) ◽  
pp. 2402-2408 ◽  
Author(s):  
B Haribabu ◽  
R P Dottin
Keyword(s):  
Gene Expression ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Cell Surface Receptor ◽  
Intracellular Camp ◽  
Response Curves ◽  
Pharmacological Characterization ◽  
Surface Receptor ◽  
Extracellular Molecules

Extracellular molecules regulate gene expression in eucaryotes. Exogenous cyclic AMP (cAMP) affects the expression of a large number of developmentally regulated genes in Dictyostelium discoideum. Here, we determine the specificity of the receptor(s) which mediates gene expression by using analogs of cAMP. The order of potency with which these analogs affect the expression of specific genes is consistent with the specificity of their binding to a cell surface receptor and is distinct from their affinity for intracellular cAMP-dependent protein kinase. Dose-response curves with cAMP and adenosine 3',5'-monophosphorothioate, a nonhydrolyzable analog, revealed that the requirement for high concentrations of exogenous cAMP for regulating gene expression is due to the rapid degradation of cAMP by phosphodiesterase. The addition of low concentrations of cAMP (100 nM) or analogs in pulses also regulates gene expression. Both the genes that are positively regulated by exogenous cAMP and the discoidin gene, which is negatively regulated, respond to cAMP analogs to the same degree. Genes expressed in prespore or prestalk cells are also similarly regulated. These data suggest that the effects are mediated through the same receptor. The specificity of this receptor is indistinguishable from that of the well-characterized cell surface cAMP receptor.

A pharmacologically distinct cyclic AMP receptor is responsible for the regulation of gp80 expression in Dictyostelium discoideum

1990 ◽  
Vol 10 (7) ◽  
pp. 3297-3306
Author(s):  
P C Ma ◽  
C H Siu
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Dictyostelium Discoideum ◽  
Surface Glycoprotein ◽  
Intracellular Camp ◽  
Cell Surface Glycoprotein ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation ◽  
Camp Receptor

The EDTA-resistant cell-cell adhesion expressed at the aggregation stage of Dictyostelium discoideum is mediated by a cell surface glycoprotein of Mr 80,000 (gp80). The expression of gp80 is developmentally regulated by cyclic AMP (cAMP). In vitro nuclear run-on experiments show that transcription of the gp80 gene is initiated soon after the onset of development. The basal level of gp80 transcription is significantly augmented by exogenous cAMP pulses. Interestingly, in analog studies, 2'-deoxy-cAMP, 8-bromo-cAMP, and N6-monobutyryl-cAMP are all capable of inducing a rapid accumulation of gp80 mRNA, suggesting the presence of a unique cAMP receptor that responds equally well to these analogs. To determine whether intracellular cAMP plays a role in the regulation of gp80 expression, caffeine was used to block cAMP-induced receptor-mediated adenylate cyclase activation. Expression of gp80 mRNA was blocked in caffeine-treated cells but could be substantially restored by treatment with exogenous cAMP pulses, suggesting that adenylate cyclase activation is not required. gp80 expression was also examined in the signal transduction mutants synag 7 and frigid A. In both mutants, gp80 was expressed at the basal level. Pulses of cAMP as well as 2'-deoxy-cAMP and N6-monobutyryl-cAMP were capable of restoring the normal level of gp80 expression in synag 7 cells. These results, taken together, indicate bimodal regulation of gp80 expression during development and the involvement of a novel cAMP receptor in the transmembrane signalling pathway that regulates gp80 gene expression.

Spike-shaped oscillations in the absence of measurable changes in cyclic AMP concentration in a mutant of Dictyostelium discoideum

1987 ◽  
Vol 87 (5) ◽  
pp. 723-730
Author(s):  
B. Wurster ◽  
R. Mohn
Keyword(s):  
Light Scattering ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Parent Strain ◽  
Reaction System ◽  
Cell Suspensions ◽  
Deficient Mutant ◽  
Cyclic Amp Synthesis

Periodic activities of Dictyostelium discoideum cells involve two types of oscillations, spike-shaped and sinusoidal. Spike-shaped oscillations are accompanied by the periodic synthesis and release of cyclic AMP, and cyclic AMP-activated cyclic AMP synthesis is believed to control these oscillations. Experiments described here call into question the importance of cyclic AMP in spike-shaped oscillations. Cell suspensions of strain agip43, an aggregation-deficient mutant of D. discoideum, displayed spike-shaped oscillations in light scattering with period lengths about 1.5 times larger than those of the parent strain. These oscillations were not accompanied by measurable oscillations of cyclic AMP and cyclic GMP. Applied cyclic AMP pulses elicited increases of two- to threefold in the cyclic AMP level and increases of seven- to ninefold in the cyclic GMP concentration. Cyclic AMP additions caused phase shifts in the oscillations of agip43 cells, suggesting that cyclic AMP receptors at the cell surface communicate with the oscillator. We interpret these results in terms of an oscillator not based on cyclic AMP. This oscillator should be coupled to the reaction system involving cyclic AMP synthesis and release. The latter can operate in an oscillatory manner in the parent strain Ax2 but not in mutant agip43.

The effects of inhibition of protein glycosylation on the aggregation of Dictyostelium discoideum

Development ◽  
1983 ◽  
Vol 78 (1) ◽  
pp. 229-248
Author(s):  
Charles John McDonald ◽  
Jeffrey Sampson
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Total Protein ◽  
Subsequent Development ◽  
Protein Glycosylation ◽  
Normal Process ◽  
Contact Sites ◽  
Cyclic Amp Phosphodiesterase

At concentrations greater than 10 µg ml−1 tunicamycin inhibited the incorporation of [3H]mannose into glycoproteins during the early phase of development in Dictyostelium discoideum, however, total protein synthesis was unaffected. Tunicamycin also interfered with the normal process of aggregation. In its presence small aggregates were observed at the time of normal aggregation, but amoebae failed to aggregate completely and subsequent development was inhibited. Inhibition of normal aggregation by tunicamycin was found to be reversible. The appearance of cell-associated and secreted cyclic AMP phosphodiesterase and cell-surface contact sites A was prevented by tunicamycin but cell surface cyclic AMP receptor activity developed normally in its presence. Tunicamycin also prevented amoebae from acquiring the ability to chemotact toward cyclic AMP. Addition of exogenous cyclic AMP phosphodiesterase restored the ability of amoebae to chemotact toward cyclic AMP in the presence of tunicamycin. Our data suggest that the primary block in aggregation caused by tunicamycin results from the inhibition of expression of active cyclic AMP phosphodiesterase.

scholarly journals Role of SP65 in Assembly of the Dictyostelium discoideum Spore Coat

2007 ◽  
Vol 6 (7) ◽  
pp. 1137-1149 ◽  
Author(s):  
Talibah Metcalf ◽  
Hanke van der Wel ◽  
Ricardo Escalante ◽  
Leandro Sastre ◽  
Christopher M. West
Keyword(s):  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Outer Layer ◽  
Fluorescent Protein ◽  
De Novo ◽  
Middle Layer ◽  
In Vivo Studies ◽  
Permeability Barrier ◽  
Spore Coat

ABSTRACT Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.

Spatial and temporal regulation of a foreign gene by a prestalk-specific promoter in transformed Dictyostelium discoideum

1986 ◽  
Vol 6 (3) ◽  
pp. 811-820
Author(s):  
S Datta ◽  
R H Gomer ◽  
R A Firtel
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Regulatory Elements ◽  
Foreign Gene ◽  
Specific Gene ◽  
Cell Type ◽  
Coding Region ◽  
Endogenous Gene ◽  
Temporal Regulation

We analyzed a developmentally regulated prestalk-specific gene from Dictyostelium discoideum encoding a cathepsin-like protease. A hybrid gene was constructed by fusing 2.5 kilobases of 5' flanking sequences and part of the coding region of the gene in-frame to the Escherichia coli beta-glucuronidase gene and was transformed into D. discoideum cells. In cells transformed with this vector, the gene fusion showed the same temporal regulation as the endogenous gene during multicellular development and, like endogenous prestalk genes, was highly inducible by cyclic AMP in in vitro cell cultures. Moreover, immunofluorescence studies showed that the fusion protein had the same spatial distribution within the migrating pseudoplasmodium as the endogenous gene. The results indicate that the regions of the D. discoideum prestalk-specific cathepsin gene contain all the necessary information for proper temporal, spatial, and cyclic AMP regulation of a prestalk cell-type gene in D. discoideum transformants and leads the way for experiments to identify the cell-type-specific regulatory elements.

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