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1.
Methods Inf Med ; 48(3): 229-35, 2009.
Article in English | MEDLINE | ID: mdl-19387502

ABSTRACT

BACKGROUND: The genetic cellular response to internal and external changes is determined by the sequence and structure of gene-regulatory promoter regions. OBJECTIVES: Using data on gene-regulatory elements (i.e., either putative or known transcription factor binding sites) and data on gene expression profiles we can discover structural elements in promoter regions and infer the underlying programs of gene regulation. Such hypotheses obtained in silico can greatly assist us in experiment planning. The principal obstacle for such approaches is the combinatorial explosion in different combinations of promoter elements to be examined. METHODS: Stemming from several state-of-the-art machine learning approaches we here propose a heuristic, rule-based clustering method that uses gene expression similarity to guide the search for informative structures in promoters, thus exploring only the most promising parts of the vast and expressively rich rule-space. RESULTS: We present the utility of the method in the analysis of gene expression data on budding yeast S. cerevisiae where cells were induced to proliferate peroxisomes. CONCLUSIONS: We demonstrate that the proposed approach is able to infer informative relations uncovering relatively complex structures in gene promoter regions that regulate gene expression.


Subject(s)
Gene Expression Regulation/genetics , Gene Expression/genetics , Promoter Regions, Genetic/genetics , Algorithms , Saccharomyces cerevisiae/genetics , Validation Studies as Topic
2.
Stud Health Technol Inform ; 84(Pt 2): 956-9, 2001.
Article in English | MEDLINE | ID: mdl-11604873

ABSTRACT

The sequencing of the human genome and the genomes of several model organisms is the first step toward the long-term objective of genetic research: the identification of all genes, and the discovery of their functions and mutual interactions. This article presents a methodology and a computer program called GenePath to support the discovery of gene function. GenePath uses mutant data and available genetic knowledge to identify potential genetic pathways. Several pilot applications based on experimental results from Dictyostelium and C. elegans confirmed the usefulness of the proposed schema. Our results suggest that GenePath is a valuable tool that can be used as an intelligent assistant to support genetic reasoning.


Subject(s)
Artificial Intelligence , Genomics/methods , Software , Animals , Caenorhabditis elegans/genetics , Computational Biology , Dictyostelium/genetics , Mutation
3.
Funct Integr Genomics ; 1(5): 279-93, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11793247

ABSTRACT

The genome of Dictyostelium discoideum is being sequenced by an international consortium and is scheduled for completion in the next few years. The sequence will accelerate research into a number of phenomena carried out by these versatile soil amoebae, providing insight into analogous processes that operate in a wide range of eukaryotes. These include the dynamic regulation of the cytoskeleton during chemotaxis, intercellular communication during multicellular development and the intracellular growth of bacterial pathogens. The current state of the genome project is summarized and the challenges of sequencing a genome with unusually low guanine and cytosine content and with a bimodal base composition distribution are discussed. The prospects for functional analyses at the genomic scale are also considered.


Subject(s)
Dictyostelium/genetics , Genome, Protozoan , Animals , Gene Expression Profiling , Mutation , Protozoan Proteins/physiology , Signal Transduction/physiology
4.
Mol Biol Cell ; 11(8): 2803-20, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10930471

ABSTRACT

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.


Subject(s)
Chemotaxis , Cyclic AMP-Dependent Protein Kinases/physiology , Dictyostelium/physiology , Protozoan Proteins , Pseudopodia/physiology , 3',5'-Cyclic-AMP Phosphodiesterases , Animals , Cell Aggregation , Computer Simulation , Cyclic AMP/physiology , Cyclic AMP-Dependent Protein Kinases/genetics , Dictyostelium/cytology , Dictyostelium/genetics , Genes, Protozoan , Mutation , Myosins/metabolism , Pseudopodia/metabolism , Signal Transduction , Transformation, Genetic
5.
J Eukaryot Microbiol ; 47(4): 334-9, 2000.
Article in English | MEDLINE | ID: mdl-11140446

ABSTRACT

Dictyostelium discoideum is a useful model for molecular studies of cell biology and development. The 34-megabase Dictyostelium genome is currently being sequenced through the efforts of an international consortium. The genome is expected to encode 8-10,000 genes, including all those required for a free-living eukaryote capable of multicellular development. A complete description of the Dictyostelium genome will open the way toward the application of genome-based experimental approaches to studies of cell biology and development in this organism, and allow detailed physiological and evolutionary comparisons to other species.


Subject(s)
Dictyostelium , Genome, Protozoan , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Animals , Dictyostelium/genetics , Dictyostelium/physiology
6.
Mol Cell Biol ; 19(7): 4750-6, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10373524

ABSTRACT

SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate. Genetic evidence indicates that the response is dependent on the dhkA gene. This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways. The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310-amino-acid loop that could bind a ligand. By inserting MYC6 epitopes into DhkA, we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic. Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100-fold-higher levels of SDF-2 than required to induce dhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope. To examine the enzymatic activity of DhkA, we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation. Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation. Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity. Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB. Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.


Subject(s)
Dictyostelium/physiology , Membrane Proteins/metabolism , Peptides , Protein Kinases/metabolism , Proteins/metabolism , Protozoan Proteins , Animals , Catalytic Domain , Cell Differentiation , Cell Membrane/enzymology , Cell Membrane/metabolism , Dictyostelium/drug effects , Dictyostelium/metabolism , Intercellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Mutagenesis, Site-Directed , Phosphorylation , Protein Kinases/genetics , Proteins/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
7.
Dev Biol ; 208(1): 1-13, 1999 Apr 01.
Article in English | MEDLINE | ID: mdl-10075837

ABSTRACT

Receptor-mediated activation of adenylyl cyclase (ACA) in Dictyostelium requires CRAC protein. Upon translocation to the membrane, this pleckstrin homology (PH) domain protein stimulates ACA and thereby mediates developmental aggregation. CRAC may also have roles later in development since CRAC-null cells can respond to chemotactic signals and participate in developmental aggregation when admixed with wild-type cells, but they do not complete development within such chimeras. To test whether the role of CRAC in postaggregative development is related to the activation of ACA, chemotactic aggregation was bypassed in CRAC-null cells by activating the cAMP-dependent protein kinase (PKA). While such strains formed mounds, they did not complete fruiting body morphogenesis or form spores. Expression of CRAC in the prespore cells of these strains rescued sporulation and fruiting body formation. This later function of CRAC does not appear to require its PH domain since the C-terminal portion of the protein (CRAC-DeltaPH) can substitute for full-length CRAC in promoting spore cell formation and morphogenesis. No detectable ACA activation was observed in any of the CRAC-null strains rescued by PKA activation and expression of CRAC-DeltaPH. Finally, we found that the development of CRAC-null ACA-null double mutants could be rescued by the activation of PKA together with the expression of CRAC-DeltaPH. Thus, there appears to be a required function for CRAC in postaggregative development that is independent of its previously described function in the ACA activation pathway.


Subject(s)
Adenylyl Cyclases/metabolism , Dictyostelium/enzymology , Phosphoproteins , Protozoan Proteins/genetics , Animals , Blood Proteins/genetics , Cell Aggregation , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cytosol/enzymology , Dictyostelium/growth & development , Enzyme Activation , Gene Expression Regulation, Developmental/genetics , Phenotype , RNA, Messenger/metabolism , Spores/metabolism
8.
Development ; 125(4): 691-9, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9435289

ABSTRACT

A cAMP-specific phosphodiesterase was found that is stimulated by binding to the regulatory subunit of cAMP-dependent protein kinase, PKA-R, from either Dictyostelium or mammals. The phosphodiesterase is encoded by the regA gene of Dictyostelium, which was recovered in a mutant screen for strains that sporulate in the absence of signals from prestalk cells. The sequence of RegA predicts that it will function as a member of a two-component system. Genetic analyses indicate that inhibition of the phosphodiesterase results in an increase in the activity of PKA, which acts at a check point for terminal differentiation. Conserved components known to affect memory, learning and differentiation in flies and vertebrates suggest that a similar circuitry functions in higher eukaryotes.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Dictyostelium/cytology , Dictyostelium/enzymology , 3',5'-Cyclic-AMP Phosphodiesterases/genetics , Animals , Dictyostelium/genetics , Genes, Fungal , Genes, Protozoan , Models, Biological , Mutation , Phenotype , Phosphorylation , Signal Transduction , Spores, Fungal/enzymology , Spores, Fungal/genetics
9.
Curr Opin Microbiol ; 1(6): 643-8, 1998 Dec.
Article in English | MEDLINE | ID: mdl-10066536

ABSTRACT

Conserved signal transduction pathways that use phosphorelay from histidine kinases through an intermediate transfer protein (H2) to response regulators have been found in a variety of eukaryotic microorganisms. Several of these pathways are linked to mitogen-activated protein kinase cascades. These networks control different physiological responses including osmoregulation, cAMP levels and cellular morphogenesis.


Subject(s)
Fungi/metabolism , Protein Kinases/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Dictyosteliida/enzymology , Dictyosteliida/genetics , Dictyosteliida/metabolism , Eukaryotic Cells , Fungi/enzymology , Fungi/genetics , Histidine Kinase , Molecular Sequence Data , Protein Kinases/chemistry , Protein Kinases/genetics , Signal Transduction/genetics
10.
Gene ; 195(2): 127-30, 1997 Aug 22.
Article in English | MEDLINE | ID: mdl-9305756

ABSTRACT

We isolated a cDNA from Dictyostelium discoideum that encodes a 30 kDa protein with significant similarity to members of the major intrinsic protein (MIP) family of membrane transporters. The most closely related protein in the public data bases is an aquaporin from Cicadella viridis which shows 34% identity. The cDNA was used to isolate and characterize genomic fragments carrying the Dictyostelium gene which we named wacA. Genomic probes were used to recognize wacA mRNA isolated at various stages of development. The results showed that the gene is developmentally regulated such that the mRNA first appears at 12 h of development and is retained throughout the remainder of development. In situ hybridization of whole mounts prepared at 15 h of development showed that wacA mRNA accumulates exclusively in prespore cells and is absent from prestalk cells. Although wacA expression is prespore specific, disruption of the gene by homologous recombination did not result in observable alterations in the formation of spores or their resistance to osmotic challenges.


Subject(s)
Aquaporins , Dictyostelium/genetics , Membrane Glycoproteins , Porins/genetics , Protozoan Proteins , Amino Acid Sequence , Animals , Chromosome Mapping , Cloning, Molecular , DNA Probes/genetics , Dictyostelium/growth & development , Eye Proteins/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Fungal , Genes, Fungal , Genes, Protozoan , In Situ Hybridization , Insecta/genetics , Molecular Sequence Data , Osmotic Pressure , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombination, Genetic , Sequence Homology, Amino Acid , Spores, Fungal/genetics , Spores, Fungal/growth & development , Spores, Fungal/metabolism
11.
Dev Biol ; 185(1): 34-41, 1997 May 01.
Article in English | MEDLINE | ID: mdl-9169048

ABSTRACT

About 8 hr after the initiation of development in Dictyostelium discoideum, a few randomly scattered cells express prestalk specific genes and subsequently sort out to the top of the aggregate where they form a tip. The tip elongates and forms the anterior of the migrating slug before differentiating into a stalk which supports the ball of spores in a mature fruiting body. Using REMI mutagenesis we isolated a mutant strain, AK244, in which the initial aggregate subdivides to give a highly papillated surface. This mutant fails to form slugs and appears to have a defect in sorting of prestalk cells. The disrupted gene, tipA, encodes a novel 83-kDa protein and is preferentially expressed in PST-O cells after the cell types have sorted out. Mutant strains that lack TipA express the prestalk-specific gene ecmA at reduced levels and form very few spores. These defects cannot be overcome by developing the mutant cells in the presence of wild-type cells. Thus, TipA acts in a cell-autonomous manner at an early stage in development. Using strains carrying reporter constructs, we found that mutant cells expressing a prestalk marker remain dispersed in the aggregates. Prespore cells appear to sort such that the base is free of cells expressing cell-type-specific markers. Even after 20 hr of development, when wild-type cells are undergoing terminal differentiation, prestalk cells in tipA mutants form very small clumps, most of which fail to sort to the periphery or the tops of aggregates. The tipA gene appears to play an essential role in the sorting of the initial cell types.


Subject(s)
Cell Lineage , Dictyostelium/genetics , Fungal Proteins/genetics , Genes, Protozoan/physiology , Protozoan Proteins , Amino Acid Sequence , Animals , Cell Adhesion , Cloning, Molecular , Dictyostelium/cytology , Dictyostelium/growth & development , Fluorescent Antibody Technique , Gene Expression Regulation, Developmental/genetics , Molecular Sequence Data , Phenotype
12.
J Cell Sci ; 110 ( Pt 10): 1141-5, 1997 May.
Article in English | MEDLINE | ID: mdl-9191038

ABSTRACT

Autophosphorylating histidine kinases are an ancient conserved family of enzymes that are found in eubacteria, archaebacteria and eukaryotes. They are activated by a wide range of extracellular signals and transfer phosphate moieties to aspartates found in response regulators. Recent studies have shown that such two-component signal transduction pathways mediate osmoregulation in Saccharomyces cerevisiae, Dictyostelium discoideum and Neurospora crassa. Moreover, they play pivotal roles in responses of Arabidopsis thaliana to ethylene and cytokinin. A transmembrane histidine kinase encoded by dhkA accumulates when Dictyostelium cells aggregate during development. Activation of DhkA results in the inhibition of its response regulator, RegA, which is a cAMP phosphodiesterase that regulates the cAMP dependent protein kinase PKA. When PKA is activated late in the differentiation of prespore cells, they encapsulate into spores. There is evidence that this two-component system participates in a feedback loop linked to PKA in prestalk cells such that the signal to initiate encapsulation is rapidly amplified. Such signal transduction pathways can be expected to be found in a variety of eukaryotic differentiations since they are rapidly reversible and can integrate disparate signals.


Subject(s)
Protein Kinases/metabolism , Signal Transduction/physiology , Animals , Arabidopsis/drug effects , Arabidopsis/enzymology , Arabidopsis/genetics , Cytokinins/pharmacology , Dictyostelium/genetics , Dictyostelium/growth & development , Dictyostelium/metabolism , Ethylenes/pharmacology , Eukaryotic Cells , Histidine Kinase , Neurospora crassa/genetics , Neurospora crassa/metabolism , Phosphorylation , Protein Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Signal Transduction/genetics , Water-Electrolyte Balance
13.
Proc Natl Acad Sci U S A ; 93(26): 15260-5, 1996 Dec 24.
Article in English | MEDLINE | ID: mdl-8986798

ABSTRACT

We have found conditions for saturation mutagenesis by restriction enzyme mediated integration that result in plasmid tagging of disrupted genes. Using this method we selected for mutations in genes that act at checkpoints downstream of the intercellular signalling system that controls encapsulation in Dictyostelium discoideum. One of these genes, mkcA, is a member of the mitogen-activating protein kinase cascade family while the other, regA is a novel bipartite gene homologous to response regulators in one part and to cyclic nucleotide phosphodiesterases in the other part. Disruption of either of these genes results in partial suppression of the block to spore formation resulting from the loss of the prestalk genes, tagB and tagC. The products of the tag genes have conserved domains of serine protease attached to ATP-driven transporters, suggesting that they process and export peptide signals. Together, these genes outline an intercellular communication system that coordinates organismal shape with cellular differentiation during development.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/genetics , Dictyostelium/physiology , Genes, Suppressor , Signal Transduction , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Conserved Sequence , Dictyostelium/genetics , Genes, Fungal , Major Histocompatibility Complex , Mammals , Molecular Sequence Data , Mutagenesis , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Plasmids , Protein Kinases/metabolism , Restriction Mapping , Sequence Homology, Amino Acid , Spores, Fungal
14.
EMBO J ; 15(15): 3890-8, 1996 Aug 01.
Article in English | MEDLINE | ID: mdl-8670894

ABSTRACT

A mutant which failed to complete development was isolated from a population of cells that had been subjected to insertional mutagenesis using restriction enzyme-mediated integration. The disrupted gene, dhkA, encodes the conserved motifs of a histidine kinase as well as the response regulator domain. It is likely that the histidine in DhkA is autophosphorylated and the phosphate passed to one or more response regulators. Such two-component systems function in a variety of bacterial signal transduction pathways and have been characterized recently in yeast and Arabidopsis. In Dictyostelium, we found that DhkA functions both in the regulation of prestalk gene expression and in the control of the terminal differentiation of prespore cells.


Subject(s)
Dictyostelium/enzymology , Protein Kinases/genetics , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Dictyostelium/genetics , Dictyostelium/growth & development , Gene Expression Regulation, Fungal , Genes, Recessive , Histidine Kinase , In Situ Hybridization , Molecular Sequence Data , Mutation , Phenotype , Phosphorylation , Plasmids/metabolism
15.
Dev Biol ; 174(2): 214-20, 1996 Mar 15.
Article in English | MEDLINE | ID: mdl-8631494

ABSTRACT

Prespore and prestalk cells can be distinguished within aggregates of Dictyostelium by the expression of well-characterized cell type-specific genes. Fusion of the tagB regulatory region to Escherichia coli beta-galactosidase revealed that this prestalk specific gene marks the differentiation of the initial prestalk cell population, PST-1. The reporter gene was expressed normally in tagB- mutant cells despite the fact that they do not accumulate measurable levels of DIF-I, a morphogen that was previously implicated in prestalk differentiation. In an independent experimental system, wild-type cells respond to the addition of DIF-I by induction of the prestalk marker ecmA and repression of the prespore marker cotB. We found that DIF-1 did not affect the expression of the tagB or carB genes, both of which are prestalk specific and essential for PST-A cell differentiation. We conclude that the initiation of prestalk development is not dependent on DIF-1 and suggest that the morphogen participates mainly at later stages.


Subject(s)
ATP-Binding Cassette Transporters , Caenorhabditis elegans Proteins , Carrier Proteins/physiology , Dictyostelium/cytology , Helminth Proteins/physiology , Proteins , Protozoan Proteins , Animals , Carrier Proteins/genetics , Cell Differentiation/genetics , Cloning, Molecular , Dictyostelium/genetics , Dictyostelium/growth & development , Escherichia coli/enzymology , Gene Expression Regulation, Developmental , Genes, Reporter , Hexanones , Hydrocarbons, Chlorinated , Recombinant Fusion Proteins/genetics , Serine Endopeptidases/genetics , beta-Galactosidase/genetics
16.
Proc Natl Acad Sci U S A ; 92(12): 5660-3, 1995 Jun 06.
Article in English | MEDLINE | ID: mdl-7777565

ABSTRACT

Dictyostelium discoideum cells initiate development when nutrients are depleted. DNA synthesis decreases rapidly thereafter but resumes during late aggregation, only in prespore cells. This observation has been previously interpreted as indicating progression of prespore cells through the cell cycle during development. We show that developmental DNA replication occurs only in mitochondria and not in nuclei. We also show that the prestalk morphogen known as differentiation-inducing factor 1 can inhibit mitochondrial respiration. A model is proposed for cell type divergence, based on competition to become prespores, that involves mitochondrial replication in prespore cells and reduction of mitochondrial activity in prestalk cells.


Subject(s)
Cell Nucleus/metabolism , DNA Replication , DNA, Mitochondrial/biosynthesis , Dictyostelium/genetics , Animals , Dictyostelium/growth & development , Dictyostelium/metabolism , Hexanones/metabolism
17.
Genes Dev ; 9(9): 1111-22, 1995 May 01.
Article in English | MEDLINE | ID: mdl-7744252

ABSTRACT

The prestalk-specific gene, tagB, was disrupted by restriction enzyme-mediated integration (REMI) mutagenesis. Mutant aggregates exhibit a cell-autonomous defect in specialization of PST-A cells, a prestalk subpopulation that forms the tip and eventually forms the stalk of the fruiting body. Cooperative (non-cell-autonomous) defects were found in sporulation and in specialization of prestalk cells that eventually form the upper cup of the fruiting body (PST-O). The pattern of ecmA::lacZ expression in mutant tagB- cells defines a primary prestalk population, PST-I, from which other prestalk cells differentiate. After PST-A cells differentiate, they induce remaining PST-I cells to become PST-O cells. Subsequently, prestalk cells induce encapsulation of prespore cells during culmination. tagB is homologous to serine protease and to multidrug resistance (MDR) transporter genes, implying a mechanism of action that includes proteolysis and export of peptide signals. Intercellular communication via TagB may mediate integration of cellular differentiation with morphogenesis.


Subject(s)
Carrier Proteins/physiology , Dictyostelium/growth & development , Dictyostelium/genetics , Drug Resistance, Multiple/genetics , Genes, Fungal/genetics , Protozoan Proteins , Serine Endopeptidases/physiology , ATP-Binding Cassette Transporters/genetics , Amino Acid Sequence , Animals , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Cell Differentiation , Cloning, Molecular , Dictyostelium/cytology , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Gene Expression Regulation, Fungal/drug effects , Hexanones/metabolism , Hexanones/pharmacology , Molecular Sequence Data , Mutagenesis, Insertional/methods , RNA, Fungal/biosynthesis , RNA, Messenger/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics
18.
J Cell Biol ; 128(3): 405-13, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7844154

ABSTRACT

Analysis of a developmental mutant in Dictyostelium discoideum which is unable to initiate morphogenesis has shown that a protein kinase of the MAP kinase/ERK family affects relay of the cAMP chemotactic signal and cell differentiation. Strains in which the locus encoding ERK2 is disrupted respond to a pulse of cAMP by synthesizing cGMP normally but show little synthesis of cAMP. Since mutant cells lacking ERK2 contain normal levels of both the cytosolic regulator of adenylyl cyclase (CRAC) and manganese-activatable adenylyl cyclase, it appears that this kinase is important for receptor-mediated activation of adenylyl cyclase.


Subject(s)
Adenylyl Cyclases/metabolism , Dictyostelium/enzymology , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Amino Acid Sequence , Animals , Enzyme Activation , GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinase 1 , Molecular Sequence Data , Mutation , Phenotype , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Sequence Homology, Amino Acid
19.
J Cell Biol ; 126(6): 1537-45, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8089184

ABSTRACT

Adenylyl cyclase in Dictyostelium, as in higher eukaryotes, is activated through G protein-coupled receptors. Insertional mutagenesis into a gene designated dagA resulted in cells that cannot activate adenylyl cyclase, but have otherwise normal responses to exogenous cAMP. Neither cAMP treatment of intact cells nor GTP gamma S treatment of lysates stimulates adenylyl cyclase activity in dagA mutants. A cytosolic protein that activates adenylyl cyclase, CRAC, has been previously identified. We trace the signaling defect in dagA- cells to the absence of CRAC, and we demonstrate that dagA is the structural gene for CRAC. The 3.2-kb dagA mRNA encodes a predicted 78.5-kD product containing a pleckstrin homology domain, in agreement with the postulated interaction of CRAC with activated G proteins. Although dagA expression is tightly developmentally regulated, the cDNA restores normal development when constitutively expressed in transformed mutant cells. In addition, the megabase region surrounding the dagA locus was mapped. We hypothesize that CRAC acts to connect free G protein beta gamma subunits to adenylyl cyclase activation. If so, it may be the first member of an important class of coupling proteins.


Subject(s)
Adenylyl Cyclases/metabolism , Dictyostelium/metabolism , Fungal Proteins/physiology , Phosphoproteins , Protozoan Proteins , Amino Acid Sequence , Animals , Base Sequence , Blood Proteins/chemistry , Cytosol/chemistry , Dictyostelium/genetics , Dictyostelium/growth & development , Enzyme Activation/physiology , Fungal Proteins/chemistry , GTP-Binding Proteins/physiology , Genes, Fungal , Molecular Sequence Data , Mutagenesis, Insertional , Receptors, Cyclic AMP/physiology , Restriction Mapping , Sequence Homology, Amino Acid , Signal Transduction/physiology
20.
Genes Dev ; 8(8): 948-58, 1994 Apr 15.
Article in English | MEDLINE | ID: mdl-7926779

ABSTRACT

Strain AK127 is a developmental mutant of Dictyostelium discoideum that was isolated by restriction enzyme-mediated integration (REMI). Mutant cells aggregate normally but are unable to proceed past the loose aggregate stage. The cloned gene, lagC (loose aggregate C), encodes a novel protein of 98 kD that contains an amino-terminal signal sequence and a putative carboxy-terminal transmembrane domain. The mutant strain AK127 shows no detectable lagC transcript upon Northern analysis, indicating that the observed phenotype is that of a null allele. Expression of the lagC cDNA in AK127 cells complements the arrest at the loose aggregate stage, indicating that the mutant phenotype results from disruption of the lagC gene. In wild-type cells, lagC mRNA is induced at the loose aggregate stage and is expressed through the remainder of development. lagC- null cells aggregate but then disaggregate and reaggregate to form small granular mounds. Mature spores are produced at an extremely low efficiency (< 0.1% of wild type), appearing only after approximately 72 hr, whereas wild-type strains produce mature spores by 26 hr. lagC- null cells accumulate reduced levels of transcripts for the prestalk-enriched genes rasD and CP2 and do not express the DIF-induced prestalk-specific gene ecmA or the cAMP-induced prespore-specific gene SP60 to significant levels. In chimeric organisms resulting from the coaggregation of lagC- null and wild-type cells, cell-type-specific gene expression is rescued in the lagC- null cells; however, lagC- prespore cells are localized to the posterior of the prespore region and do not form mature spores, suggesting that LagC protein has both no cell-autonomous and cell-autonomous functions. Overexpression of lagC from an actin promoter in both wild-type and lagC- cells causes a delay at the tight aggregate stage, the first stage requiring LagC activity. These results suggest that the LagC protein functions as a nondiffusible cell-cell signaling molecule that is required for multicellular development.


Subject(s)
Cell Communication/genetics , Dictyostelium/growth & development , Genes, Protozoan/genetics , Membrane Proteins/genetics , Protozoan Proteins , Signal Transduction/genetics , Actins/biosynthesis , Actins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Differentiation/genetics , DNA, Complementary/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Dictyostelium/cytology , Dictyostelium/genetics , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Histocytochemistry , Membrane Proteins/biosynthesis , Molecular Sequence Data , Morphogenesis/genetics , Mosaicism , Mutation , RNA, Messenger/biosynthesis , RNA-Binding Proteins , Recombinant Fusion Proteins , Sequence Analysis, DNA , Transcription Factors/biosynthesis , Transcription Factors/genetics , ras Proteins/biosynthesis , ras Proteins/genetics
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