An adenylyl cyclase that functions during late development of Dictyostelium

Development ◽  
1999 ◽  
Vol 126 (23) ◽  
pp. 5463-5471 ◽  
Author(s):  
F. Soderbom ◽  
C. Anjard ◽  
N. Iranfar ◽  
D. Fuller ◽  
W.F. Loomis
Keyword(s):  
Adenylyl Cyclase ◽  
Critical Role ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Adenylyl Cyclase Activity ◽  
Extracellular Signals ◽  
Mutant Strains ◽  
Morphological Mutants ◽  
Low Levels ◽  
Mutant Cells

A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(−) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(−) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(−) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding

1993 ◽  
Vol 13 (7) ◽  
pp. 4087-4097
Author(s):  
J Wang ◽  
N Suzuki ◽  
Y Nishida ◽  
T Kataoka
Keyword(s):  
Heat Shock ◽  
Adenylyl Cyclase ◽  
Gene Replacement ◽  
Yeast Cells ◽  
Cyclase Activity ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

scholarly journals An Adenylyl Cyclase, CyaB, Acts as an Osmosensor in Myxococcus xanthus

2005 ◽  
Vol 187 (10) ◽  
pp. 3593-3598 ◽  
Author(s):  
Yoshio Kimura ◽  
Mika Ohtani ◽  
Kaoru Takegawa
Keyword(s):  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Growth Retardation ◽  
Spore Germination ◽  
Germination Rate ◽  
Myxococcus Xanthus ◽  
Receptor Type ◽  
Wild Type ◽  
Vegetative Cells ◽  
Mutant Cells

ABSTRACT We have previously reported that a receptor-type adenylyl cyclase (CyaA) of Myxococcus xanthus undergoes an osmosensor mainly during spore germination (Y. Kimura et al., J. Bacteriol. 184:3578-3585, 2002). In the present study, we cloned another receptor-type adenylyl cyclase gene (cyaB) and characterized the function of the cyaB-encoded protein. Disruption of cyaB generates a mutant that showed growth retardation at high ionic (NaCl) or high nonionic (sucrose) osmolarity. When vegetative cells were stimulated with 0.15 M NaCl, the increases in intracellular cyclic AMP levels of cyaB mutant cells were lower than those of wild-type cells. Under nonionic osmostress, the cyaB mutant exhibited reduced spore germination; however, the germination rate of the cyaB mutant was significantly higher than that of the cyaA mutant.

Requirements for the adenylyl cyclases in the development of Dictyostelium

Development ◽  
2001 ◽  
Vol 128 (18) ◽  
pp. 3649-3654 ◽  
Author(s):  
Christophe Anjard ◽  
Fredrik Söderbom ◽  
William F. Loomis
Keyword(s):  
Adenylyl Cyclase ◽  
Intracellular Signaling ◽  
Adenylyl Cyclases ◽  
Fruiting Bodies ◽  
Dependent Protein ◽  
A Cell ◽  
Express Cell ◽  
Cell Type Specific ◽  
Cell Densities ◽  
Mutant Cells

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA− mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA− PKA-Cover strain. When developed at high cell densities, acrA−acaA− PKA-Cover cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.

Regulation by protein-tyrosine phosphatase PTP2 is distinct from that by PTP1 during Dictyostelium growth and development

1994 ◽  
Vol 14 (8) ◽  
pp. 5154-5164
Author(s):  
P K Howard ◽  
M Gamper ◽  
T Hunter ◽  
R A Firtel
Keyword(s):  
Amino Acid ◽  
Protein Tyrosine Phosphatase ◽  
Growth And Development ◽  
Catalytic Domain ◽  
Tyrosine Phosphatase ◽  
Amino Acid Identity ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Acid Identity ◽  
Mutant Strains

We have cloned a gene encoding a second Dictyostelium discoideum protein-tyrosine phosphatase (PTP2) whose catalytic domain has approximately 30 to 39% amino acid identity with those of other PTPs and a 41% amino acid identity with D. discoideum PTP1. Like PTP1, PTP2 is a nonreceptor PTP with the catalytic domain located at the C terminus of the protein. PTP2 has a predicted molecular weight of 43,000 and possesses an acidic 58-amino-acid insertion 24 amino acids from the N terminus of the conserved catalytic domain. PTP2 transcripts are expressed at moderate levels in vegetative cells and are induced severalfold at the onset of development. Studies with a PTP2-lacZ reporter gene fusion indicate that PTP2, like PTP1, is preferentially expressed in prestalk and anterior-like cell types during the multicellular stages of development. PTP2 gene disruptants (ptp2 null cells) are not detectably altered in growth and show a temporal pattern of development similar to that of wild-type cells. ptp2 null slugs and fruiting bodies, however, are significantly larger than those of wild-type slugs, suggesting a role for PTP2 in regulating multicellular structures. D. discoideum strains overexpressing PTP2 from the PTP2 promoter exhibit growth rate and developmental abnormalities, the severity of which corresponds to the level of PTP2 overexpression. Strains with high overexpression of the PTP2 gene grow slowly on bacterial lawns and produce small cells in axenic medium. When development is initiated in these strains, cells are able to aggregate but then stop further morphogenesis for 6 to 8 h, after which time a variable fraction of these aggregates continue with normal timing, producing diminutive fruiting bodies. These disruption and overexpression phenotypes for PTP2 are distinct from the corresponding mutant PTP1 phenotypes. Immunoprobing PTP2 mutant strains during growth and development with antiphosphotyrosine antibodies reveals several changes in the tyrosine phosphorylation of proteins in PTP2 mutant strains compared with that in wild-type cells. These changes are different from those identified in the previously characterized corresponding PTP1 disruption and overexpression mutant strains. Thus, although PTP2 and PTP1 are nonreceptor PTPs with similar spatial patterns of expression, our findings suggest that they possess distinct regulatory functions in controlling D. discoideum growth and development.

scholarly journals The Drosophila Ortholog of MLL3 and MLL4 , trithorax related , Functions as a Negative Regulator of Tissue Growth

2013 ◽  
Vol 33 (9) ◽  
pp. 1702-1710 ◽  
Author(s):  
Hiroshi Kanda ◽  
Alexander Nguyen ◽  
Leslie Chen ◽  
Hideyuki Okano ◽  
Iswar K. Hariharan
Keyword(s):  
Histone H3 ◽  
Tissue Growth ◽  
Negative Regulator ◽  
Cyclin B ◽  
Wild Type ◽  
H3k4 Methylation ◽  
Content Type ◽  
Promote Cell Survival ◽  
Low Levels ◽  
Mutant Cells

The human MLL genes ( MLL1 to MLL4 ) and their Drosophila orthologs, trithorax ( trx ) and trithorax related ( trr ), encode proteins capable of methylating histone H3 on lysine 4. MLL1 and MLL2 are most similar to trx , while MLL3 and MLL4 are more closely related to trr . Several MLL genes are mutated in human cancers, but how these proteins regulate cell proliferation is not known. Here we show that trr mutant cells have a growth advantage over their wild-type neighbors and display changes in the levels of multiple proteins that regulate growth and cell division, including Notch, Capicua, and cyclin B. trr mutant clones display markedly reduced levels of H3K4 monomethylation without obvious changes in the levels of H3K4 di- and trimethylation. The trr mutant phenotype resembles that of Utx , which encodes a H3K27 demethylase, consistent with the observation that Trr and Utx are found in the same protein complex. In contrast to the overgrowth displayed by trr mutant tissue, trx clones are underrepresented, express low levels of the antiapoptotic protein Diap1, and exhibit only modest changes in global levels of H3K4 methylation. Thus, in Drosophila eye imaginal discs, Trr, likely functioning together with Utx, restricts tissue growth. In contrast, Trx appears to promote cell survival.

Developmentally related changes in the production and expression of endo-β-1,4-glucanases in Aspergillus nidulans

Genome ◽  
1989 ◽  
Vol 32 (2) ◽  
pp. 288-292 ◽  
Author(s):  
P. S. Bagga ◽  
S. Sharma ◽  
D. K. Sandhu
Keyword(s):  
Aspergillus Nidulans ◽  
The Other ◽  
Wild Type ◽  
Stages Of Development ◽  
Experimental Conditions ◽  
The Third ◽  
Developmental Mutants ◽  
Mutant Strains ◽  
In The Wild ◽  
Low Levels

The production and electrophoretic expression of endoglucanase(s) were compared in the wild-type and three developmental mutants of Aspergillus nidulans. In the wild type, the production of endoglucanase and its distribution in extracellular and intracellular fractions varied with the age of the culture and the yield was better in stable cultures (production of conidia and cleistothecia) as compared with shake cultures (vegetative hyphae only). Two developmental mutants, aco-T69 and aco-40, which lack the development of conidia and cleistothecia, produced low levels of endoglucanase enzymes as compared with the wild type grown under similar conditions. On the other hand, in aco-90, a mutant capable of producing cleistothecia but no conidia, endoglucanase production was better. The results indicate a correlation between cleistothecial development and endoglucanase level. The electrophoretic studies revealed the presence of three forms of endoglucanase, i.e., EGI, EGII, and EGIII. The first two were detectable in the wild type as well as in mutant strains when grown under various experimental conditions and at all the stages of development. However, the third form could be observed only during cleistothecial development, indicating that this isozyme is developmentally regulated.Key words: endoglucanases, development, Aspergillus nidulans.

scholarly journals Regulation by protein-tyrosine phosphatase PTP2 is distinct from that by PTP1 during Dictyostelium growth and development.

1994 ◽  
Vol 14 (8) ◽  
pp. 5154-5164 ◽  
Author(s):  
P K Howard ◽  
M Gamper ◽  
T Hunter ◽  
R A Firtel
Keyword(s):  
Amino Acid ◽  
Protein Tyrosine Phosphatase ◽  
Growth And Development ◽  
Catalytic Domain ◽  
Tyrosine Phosphatase ◽  
Amino Acid Identity ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Acid Identity ◽  
Mutant Strains

We have cloned a gene encoding a second Dictyostelium discoideum protein-tyrosine phosphatase (PTP2) whose catalytic domain has approximately 30 to 39% amino acid identity with those of other PTPs and a 41% amino acid identity with D. discoideum PTP1. Like PTP1, PTP2 is a nonreceptor PTP with the catalytic domain located at the C terminus of the protein. PTP2 has a predicted molecular weight of 43,000 and possesses an acidic 58-amino-acid insertion 24 amino acids from the N terminus of the conserved catalytic domain. PTP2 transcripts are expressed at moderate levels in vegetative cells and are induced severalfold at the onset of development. Studies with a PTP2-lacZ reporter gene fusion indicate that PTP2, like PTP1, is preferentially expressed in prestalk and anterior-like cell types during the multicellular stages of development. PTP2 gene disruptants (ptp2 null cells) are not detectably altered in growth and show a temporal pattern of development similar to that of wild-type cells. ptp2 null slugs and fruiting bodies, however, are significantly larger than those of wild-type slugs, suggesting a role for PTP2 in regulating multicellular structures. D. discoideum strains overexpressing PTP2 from the PTP2 promoter exhibit growth rate and developmental abnormalities, the severity of which corresponds to the level of PTP2 overexpression. Strains with high overexpression of the PTP2 gene grow slowly on bacterial lawns and produce small cells in axenic medium. When development is initiated in these strains, cells are able to aggregate but then stop further morphogenesis for 6 to 8 h, after which time a variable fraction of these aggregates continue with normal timing, producing diminutive fruiting bodies. These disruption and overexpression phenotypes for PTP2 are distinct from the corresponding mutant PTP1 phenotypes. Immunoprobing PTP2 mutant strains during growth and development with antiphosphotyrosine antibodies reveals several changes in the tyrosine phosphorylation of proteins in PTP2 mutant strains compared with that in wild-type cells. These changes are different from those identified in the previously characterized corresponding PTP1 disruption and overexpression mutant strains. Thus, although PTP2 and PTP1 are nonreceptor PTPs with similar spatial patterns of expression, our findings suggest that they possess distinct regulatory functions in controlling D. discoideum growth and development.

scholarly journals Developmental Aggregation of Myxococcus xanthus Requires frgA, anfrz-Related Gene

2000 ◽  
Vol 182 (23) ◽  
pp. 6614-6621 ◽  
Author(s):  
Kyungyun Cho ◽  
Anke Treuner-Lange ◽  
Kathleen A. O'Connor ◽  
David R. Zusman
Keyword(s):  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
Complex Life Cycle ◽  
Fruiting Bodies ◽  
Related Gene ◽  
Wild Type ◽  
Fruiting Body Formation ◽  
Body Formation ◽  
Extracellular Signals ◽  
Directed Motility

ABSTRACT Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle that includes multicellular fruiting body formation. Frizzy mutants are characterized by the formation of tangled filaments instead of hemispherical fruiting bodies on fruiting agar. Mutations in the frz genes have been shown to cause defects in directed motility, which is essential for both vegetative swarming and fruiting body formation. In this paper, we report the discovery of a new gene, called frgA (forfrz-related gene), which confers a subset of the frizzy phenotype when mutated. The frgA null mutant showed reduced swarming and the formation of frizzy aggregates on fruiting agar. However, this mutant still displayed directed motility in a spatial chemotaxis assay, whereas the majority offrz mutants fail to show directed movements in this assay. Furthermore, the frizzy phenotype of the frgA mutant could be complemented extracellularly by wild-type cells or strains carrying non-frz mutations. The phenotype of the frgAmutant is similar to that of the abcA mutant and suggests that both of these mutants could be defective in the production or export of extracellular signals required for fruiting body formation rather than in the sensing of such extracellular signals. ThefrgA gene encodes a large protein of 883 amino acids which lacks homologues in the databases. The frgA gene is part of an operon which includes two additional genes, frgBand frgC. The frgB gene encodes a putative histidine protein kinase, and the frgC gene encodes a putative response regulator. The frgB and frgCnull mutants, however, formed wild-type fruiting bodies.

scholarly journals Salmonella enterica Serovar Typhimurium NiFe Uptake-Type Hydrogenases Are Differentially Expressed In Vivo

2008 ◽  
Vol 76 (10) ◽  
pp. 4445-4454 ◽  
Author(s):  
Andrea L. Zbell ◽  
Susan E. Maier ◽  
Robert J. Maier
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Acid Stress ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Serovar Typhimurium ◽  
Low Levels ◽  
And Function ◽  
Mutant Cells

ABSTRACT Salmonella enterica serovar Typhimurium, a common enteric pathogen, possesses three NiFe uptake-type hydrogenases. The results from mouse infection studies suggest that the H2 oxidation capacity provided by these hydrogenases is important for virulence. Since the three enzymes are similar in structure and function, it may be expected that they are utilized under different locations and times during an infection. A recombination-based method to examine promoter activity in vivo (RIVET) was used to determine hydrogenase gene expression in macrophages, polymorphonuclear leukocyte (PMN)-like cells, and a mouse model of salmonellosis. The hyd and hya promoters showed increased expression in both murine macrophages and human PMN-like cells compared to that in the medium-only controls. Quantitative reverse transcription-PCR results suggested that hyb is also expressed in phagocytes. A nonpolar hya mutant was compromised for survival in macrophages compared to the wild type. This may be due to lower tolerance to acid stress, since the hya mutant was much more acid sensitive than the wild type. In addition, hya mutant cells were internalized by macrophages the same as wild-type cells. Mouse studies (RIVET) indicate that hyd is highly expressed in the liver and spleen early during infection but is expressed poorly in the ileum in infected animals. Late in the infection, the hyd genes were expressed at high levels in the ileum as well as in the liver and spleen. The hya genes were expressed at low levels in all locations tested. These results suggest that the hydrogenases are used to oxidize hydrogen in different stages of an infection.

scholarly journals Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

1993 ◽  
Vol 13 (7) ◽  
pp. 4087-4097 ◽  
Author(s):  
J Wang ◽  
N Suzuki ◽  
Y Nishida ◽  
T Kataoka
Keyword(s):  
Heat Shock ◽  
Adenylyl Cyclase ◽  
Gene Replacement ◽  
Yeast Cells ◽  
Cyclase Activity ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

Sign in / Sign up

Export Citation Format

Share Document