scholarly journals Overexpression of an activated rasG gene during growth blocks the initiation of Dictyostelium development.

1996 ◽  
Vol 16 (8) ◽  
pp. 4156-4162 ◽  
Author(s):  
M Khosla ◽  
G B Spiegelman ◽  
G Weeks
Keyword(s):  
Growth Rate ◽  
Cell Growth ◽  
Cyclic Amp ◽  
Dominant Negative ◽  
Differentiation Process ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Ras Protein ◽  
Initial Generation ◽  
Type Strains

Transformants that expressed either the wild-type rasG gene, an activated rasG-G12T gene, or a dominant negative rasG-S17N gene, all under the control of the folate-repressible discoidin (dis1gamma) promoter, were isolated. All three transformants expressed high levels of Ras protein which were reduced by growth in the presence of folate. All three transformants grew slowly, and the reduction in growth rate correlated with the amount of RasG protein produced, suggesting that RasG is important in regulating cell growth. The pVEII-rasG transformant containing the wild-type rasG gene developed normally despite the presence of high levels of RasG throughout development. This result indicates that the down regulation of rasG that normally occurs during aggregation of wild-type strains is not essential for the differentiation process. Dictyostelium transformants expressing the dominant negative rasG-S17N gene also differentiated normally. Dictyostelium transformants that overexpressed the activated rasG-G12T gene did not aggregate. The defect occurred very early in development, since the expression of car1 and pde, genes that are normally induced soon after the initiation of development, was repressed. However, when the transformant cells were pulsed with cyclic AMP, expression of both genes returned to wild-type levels. The transformants exhibited chemotaxis to cyclic AMP, and development was synergized by mixing with wild-type cells. Furthermore, cells that were pulsed with cyclic AMP for 4 h before being induced to differentiate by plating on filters produced small, but otherwise normal, fruiting bodies. These results suggest that the rasG-G12T transformants are defective in cyclic AMP production and that RasG - GTP blocks development by interfering with the initial generation of cyclic AMP pulses.

Action of a slowly hydrolysable cyclic AMP analogue on developing cells of Dictyostelium discoideum

1979 ◽  
Vol 35 (1) ◽  
pp. 321-338
Author(s):  
C. Rossier ◽  
G. Gerisch ◽  
D. Malchow
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Agar Plate ◽  
Cell Aggregation ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Camp Analogue ◽  
Order Of Magnitude

Adenosine 3′,5′-cyclic phosphorothioate (cAMP-S) is a cyclic AMP (cAMP) analogue which is only slowly hydrolysed by phosphodiesterases of Dictyostelium discoideum. The affinity of cAMP-S to cAMP receptors at the cell surface is only one order of magnitude lower than that of cAMP. cAMP-S can replace cAMP as a stimulant with respect to all receptor-mediated responses tested, including chemotaxis and the induction of cAMP pulses. cAMP-S does not affect growth of D. discoideum but it blocks cell aggregation at a uniform concentration of 5 × 10(−7) M in agar plate cultures of strain NC-4 as well as its axenically growing derivative, Ax-2. Another wild-type strain of D. discoideum, v-12, is able to aggregate on agar plates supplemented with 1 mM cAMP-S. The development of Polysphondylium pallidum and P. violaceum is also highly cAMP-S resistant. In Ax-2 both differentiation from the growth phase to the aggregation-competent stage and chemotaxis are cAMP-S sensitive, whereas in v-12 only chemotaxis is inhibited. v-12 can still form streams of cohering cells and fruiting bodies when chemotaxis is inhibited by cAMP-S. Whereas cAMP induces differentiation into stalk cells at concentrations of 10(−3) or 10(−4) M, cAMP-S has the same effect in strain v-12 at the much lower concentration of 10(−6) M.

scholarly journals Barrier Activity in Candida albicans Mediates Pheromone Degradation and Promotes Mating

2007 ◽  
Vol 6 (6) ◽  
pp. 907-918 ◽  
Author(s):  
Dana Schaefer ◽  
Pierre Côte ◽  
Malcolm Whiteway ◽  
Richard J. Bennett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Cell Growth ◽  
Cell Fusion ◽  
Aspartyl Protease ◽  
Wild Type ◽  
Pheromone Activity ◽  
Important Regulator ◽  
Mutant Cells ◽  
Type Strains

ABSTRACT Mating in Candida albicans and Saccharomyces cerevisiae is regulated by the secretion of peptide pheromones that initiate the mating process. An important regulator of pheromone activity in S. cerevisiae is barrier activity, involving an extracellular aspartyl protease encoded by the BAR1 gene that degrades the alpha pheromone. We have characterized an equivalent barrier activity in C. albicans and demonstrate that the loss of C. albicans BAR1 activity results in opaque a cells exhibiting hypersensitivity to alpha pheromone. Hypersensitivity to pheromone is clearly seen in halo assays; in response to alpha pheromone, a lawn of C. albicans Δbar1 mutant cells produces a marked zone in which cell growth is inhibited, whereas wild-type strains fail to show halo formation. C. albicans mutants lacking BAR1 also exhibit a striking mating defect in a cells, but not in α cells, due to overstimulation of the response to alpha pheromone. The block to mating occurs prior to cell fusion, as very few mating zygotes were observed in mixes of Δbar1 a and α cells. Finally, in a barrier assay using a highly pheromone-sensitive strain, we were able to demonstrate that barrier activity in C. albicans is dependent on Bar1p. These studies reveal that a barrier activity to alpha pheromone exists in C. albicans and that the activity is analogous to that caused by Bar1p in S. cerevisiae.

Evidence that elevated intracellular cyclic AMP maturation in Dictyostelium

Development ◽  
1989 ◽  
Vol 105 (4) ◽  
pp. 753-759 ◽  
Author(s):  
R.R. Kay
Keyword(s):  
Cyclic Amp ◽  
Fruiting Body ◽  
Normal Development ◽  
Environmental Influences ◽  
Intracellular Camp ◽  
Spore Coat ◽  
Transduction Pathway ◽  
Wild Type ◽  
Type Strains ◽  
Spore Maturation

Spore maturation occurs during normal development in when environmental influences induce a migrating slug a fruiting body. As the amoeboid prespore cells turn spores there is a burst of enzyme accumulation, epimerase, and at a later stage the exocytosis of of the spore coat. Evidence is presented here that triggered by an elevated intracellular cAMP number of rapidly developing (rde) mutants, whose cAMP been investigated previously, are shown to be able to submerged monolayers, whereas wild-type strains are of these mutants are best explained by a derepression transduction pathway utilizing intracellular cAMP. direct, it is shown that the permeant cAMP analogues

A dominant negative erythropoietin (EPO) receptor inhibits EPO-dependent growth and blocks F-gp55-dependent transformation

1994 ◽  
Vol 14 (4) ◽  
pp. 2257-2265
Author(s):  
D L Barber ◽  
J C DeMartino ◽  
M O Showers ◽  
A D D'Andrea
Keyword(s):  
Cell Growth ◽  
Adaptor Protein ◽  
Erythropoietin Receptor ◽  
Dominant Negative ◽  
Friend Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Efficient Expression ◽  
Virus Transformation ◽  
Dependent Growth

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated to signal cell growth by binding either EPO or F-gp55, the Friend spleen focus-forming virus glycoprotein. Activation by F-gp55 results in constitutive EPO-R signalling and the first stage of Friend virus-induced erythroleukemia. We have generated a truncated form of the EPO-R polypeptide [EPO-R(T)] which lacks the critical cytoplasmic signal-transducing domain of the EPO-R required for EPO- or F-gp55-induced cell growth. EPO-R(T) specifically inhibited the EPO-dependent growth of EPO-R-expressing Ba/F3 cells without changing the interleukin-3-dependent growth of these cells. In addition, Ba/F3 cells that coexpressed wild-type EPO-R and EPO-R(T) were resistant to transformation by F-gp55 despite efficient expression of the F-gp55 transforming oncoprotein in infected cells. EPO-R(T) inhibited the EPO-dependent tyrosine phosphorylation of wild-type EPO-R, the tyrosine kinase (JAK2), and the SH2 adaptor protein (Shc). In conclusion, the EPO-R(T) polypeptide is a dominant negative polypeptide which specifically interferes with the early stages of EPO-R-mediated signal transduction and which prevents Friend virus transformation of erythroblasts.

Modulating action of cyclic AMP on the expression of two SOS genes in Escherichia coli K-12

1987 ◽  
Vol 33 (8) ◽  
pp. 704-708 ◽  
Author(s):  
Jordi Barbé ◽  
Isidre Gibert ◽  
Ricardo Guerrero
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Cultural Conditions ◽  
Gene Coding ◽  
K 12 ◽  
Type Strains ◽  
Lambda Prophage

Ultraviolet irradiation and cyclic AMP treatment produce a synergistic effect on the induction of the clel gene (coding for bacteriocin ColE1) in wild-type strains of Escherichia coli. On the other hand, cyclic AMP does not affect the uv-mediated induction of the recA, sfiA, and umuDC genes. Growth in the presence of glucose or glycerol does not affect the factor of amplification of the expression of the clel gene in uv-irradiated cells of the wild-type strain. Although, in cultures not treated with uv, the basal level of clel induction is about twice as high in cells grown with glycerol as in those using glucose as carbon source. In recA mutants neither simultaneous nor separate treatments with either cyclic AMP or uv irradiation induced transcription of the clel gene. Moreover, cyclic AMP induced a slight increase in clel gene expression in uv-irradiated cya strains, but not in the crp mutants. Nevertheless, the pattern of the uv-mediated induction of other SOS genes, such as umuDC, was the same in the cya and crp mutants, as in their parental wild-type strains. Furthermore, the uv-mediated induction of lambda prophage was decreased after either addition of cyclic AMP or growth in cultural conditions where the level of this nucleotide was low.

scholarly journals High-throughput identification of dominant negative polypeptides in yeast

2018 ◽  
Author(s):  
Michael W. Dorrity ◽  
Christine Queitsch ◽  
Stanley Fields
Keyword(s):  
Cell Growth ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Dominant Negative ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Dna Libraries ◽  
Yeast Genes

AbstractDominant negative polypeptides can act as inhibitors by binding to the wild type protein or by titrating an essential ligand. Here, we use high-throughput sequencing of DNA libraries composed of fragments of yeast genes to identify dominant negative polypeptides based on their depletion during cell growth. The method uncovers numerous inhibitory polypeptides for a protein and thus is capable of defining interacting domains with exquisite resolution, even pinpointing individual residues that contact ligands.

Expression of a mutated ras gene in Dictyostelium discoideum alters the binding of cyclic AMP to its chemotactic receptor

1988 ◽  
Vol 90 (4) ◽  
pp. 701-706
Author(s):  
M.E. Luderus ◽  
C.D. Reymond ◽  
P.J. Van Haastert ◽  
R. Van Driel
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Cell Types ◽  
Ras Proteins ◽  
Wild Type ◽  
Ras Gene ◽  
Physiological Level ◽  
Ras Protein ◽  
Type Gene

Dictyostelium discoideum cells contain a ras gene that codes for a polypeptide that is highly homologous to the human ras proteins. Extra copies of the wild-type gene or a gene carrying a missense mutation in codon 12 (ras-Gly12 and ras-Thr12, respectively) have been introduced into Dictyostelium cells by transformation. We have investigated the properties of the chemotactic cell surface cyclic AMP receptor in crude membrane preparations of wild-type Dictyostelium cells and ras-Gly12 and ras-Thr12 transformants. In vitro, an ATP- and Ca2+-dependent reduction of the number of cyclic AMP receptors was observed in membranes from all three cell types. The number of available receptors was decreased maximally by about 50%. In the presence of ATP the half-maximal Ca2+ concentration required for this process was about 10(−5) M in wild-type and ras-Gly12 membranes, and less than 10(−7) M in ras-Thr12 membranes. Addition of GTP (but not GDP) or the phorbol ester PMA (phorbol-12-myristate-13-acetate) reduced the Ca2+ requirement of the process in wild-type and ras-Gly12 membranes to the physiological level of less than 10(−7) M. In membranes derived from ras-Thr12 cells addition of GTP or PMA had no effect. The results indicate that D. discoideum cells contain a cyclic AMP receptor-controlling pathway that can be activated in vitro and involves a GTP-binding protein and a Ca2+ plus ATP-dependent activity, possibly protein kinase C. It is concluded that the ras protein specifically interacts with this pathway; the pathway appears to be constitutively activated by the mutated ras gene product.

scholarly journals Dunce mutants of Drosophila melanogaster: mutants defective in the cyclic AMP phosphodiesterase enzyme system.

1981 ◽  
Vol 90 (1) ◽  
pp. 101-107 ◽  
Author(s):  
R L Davis ◽  
J A Kiger
Keyword(s):  
Enzyme Activity ◽  
Drosophila Melanogaster ◽  
Cyclic Amp ◽  
Enzyme System ◽  
Residual Enzyme Activity ◽  
Wild Type ◽  
Camp Content ◽  
Mutant Strains ◽  
Normal Enzyme ◽  
Type Strains

The cyclic AMP and cyclic GMP phosphodiesterase activities present in flies of six mutant strains of the dunce gene and in the parent wild-type strains are characterized. All of the mutants exhibit aberrant cyclic AMP metabolism. The mutant strains dunceM14, dunceM11, and dunceML appear to be amorphic, because they completely lack the cAMP-specific phosphodiesterase normally present in adult flies. These strains exhibit extremely high levels of cAMP. The mutant strains dunce1, dunce2, and dunceCK are hypomorphic and exhibit reduced levels of the cAMP-specific phosphodiesterase. These strains exhibit less marked increases in cAMP content compared with the three amorphic strains. The dunce2 strain possesses a residual enzyme activity that exhibits anomalous kinetics compared with those of the normal enzyme. The possibility that the dunce locus is the structural gene for the cAMP-specific phosphodiesterase is discussed.

scholarly journals A dominant negative erythropoietin (EPO) receptor inhibits EPO-dependent growth and blocks F-gp55-dependent transformation.

1994 ◽  
Vol 14 (4) ◽  
pp. 2257-2265 ◽  
Author(s):  
D L Barber ◽  
J C DeMartino ◽  
M O Showers ◽  
A D D'Andrea
Keyword(s):  
Cell Growth ◽  
Adaptor Protein ◽  
Erythropoietin Receptor ◽  
Dominant Negative ◽  
Friend Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Efficient Expression ◽  
Virus Transformation ◽  
Dependent Growth

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated to signal cell growth by binding either EPO or F-gp55, the Friend spleen focus-forming virus glycoprotein. Activation by F-gp55 results in constitutive EPO-R signalling and the first stage of Friend virus-induced erythroleukemia. We have generated a truncated form of the EPO-R polypeptide [EPO-R(T)] which lacks the critical cytoplasmic signal-transducing domain of the EPO-R required for EPO- or F-gp55-induced cell growth. EPO-R(T) specifically inhibited the EPO-dependent growth of EPO-R-expressing Ba/F3 cells without changing the interleukin-3-dependent growth of these cells. In addition, Ba/F3 cells that coexpressed wild-type EPO-R and EPO-R(T) were resistant to transformation by F-gp55 despite efficient expression of the F-gp55 transforming oncoprotein in infected cells. EPO-R(T) inhibited the EPO-dependent tyrosine phosphorylation of wild-type EPO-R, the tyrosine kinase (JAK2), and the SH2 adaptor protein (Shc). In conclusion, the EPO-R(T) polypeptide is a dominant negative polypeptide which specifically interferes with the early stages of EPO-R-mediated signal transduction and which prevents Friend virus transformation of erythroblasts.

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