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1.
Br J Pharmacol ; 171(10): 2659-70, 2014 May.
Article in English | MEDLINE | ID: mdl-24116661

ABSTRACT

BACKGROUND AND PURPOSE: Identifying and characterizing potential new therapeutic agents to target cell proliferation may provide improved treatments for neoplastic disorders such as cancer and polycystic diseases. EXPERIMENTAL APPROACH: We used the simple, tractable biomedical model Dictyostelium to investigate the molecular mechanism of naringenin, a dietary flavonoid with antiproliferative and chemopreventive actions in vitro and in animal models of carcinogenesis. We then translated these results to a mammalian kidney model, Madin-Darby canine kidney (MDCK) tubule cells, grown in culture and as cysts in a collagen matrix. KEY RESULTS: Naringenin inhibited Dictyostelium growth, but not development. Screening of a library of random gene knockout mutants identified a mutant lacking TRPP2 (polycystin-2) that was resistant to the effect of naringenin on growth and random cell movement. TRPP2 is a divalent transient receptor potential cation channel, where mutations in the protein give rise to type 2 autosomal dominant polycystic kidney disease (ADPKD). Naringenin inhibited MDCK cell growth and inhibited cyst growth. Knockdown of TRPP2 levels by siRNA in this model conferred partial resistance to naringenin such that cysts treated with 3 and 10 µM naringenin were larger following TRPP2 knockdown compared with controls. Naringenin did not affect chloride secretion. CONCLUSIONS AND IMPLICATIONS: The action of naringenin on cell growth in the phylogenetically diverse systems of Dictyostelium and mammalian kidney cells, suggests a conserved effect mediated by TRPP2 (polycystin-2). Further studies will investigate naringenin as a potential new therapeutic agent in ADPKD.


Subject(s)
Antiprotozoal Agents/pharmacology , Cell Proliferation/drug effects , Dictyostelium/drug effects , Flavanones/pharmacology , Kidney/drug effects , Polycystic Kidney, Autosomal Dominant/metabolism , Protozoan Proteins/drug effects , TRPP Cation Channels/drug effects , Animals , Dictyostelium/genetics , Dictyostelium/growth & development , Dictyostelium/metabolism , Dogs , Dose-Response Relationship, Drug , Kidney/metabolism , Kidney/pathology , Madin Darby Canine Kidney Cells , Mutation , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/pathology , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA Interference , TRPP Cation Channels/genetics , TRPP Cation Channels/metabolism , Time Factors , Transfection
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.
Dev Biol ; 220(1): 53-61, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10720430

ABSTRACT

We have identified a cellular efflux pump, RhT, with the properties of an MDR transporter-a type of ATP-binding cassette transporter whose substrates include small hydrophobic molecules. RhT transports rhodamine 123 (Rh123) and is inhibited by low temperature, energy poisons, and several MDR transport inhibitors, such as verapamil. All vegetative cells have RhT activity, but during development prestalk cells lose RhT activity while prespore cells retain it. We also identified several RhT inhibitors. The most effective inhibitor is the stalk cell-inducing chlorinated alkyl phenone, DIF-1. The RhT inhibitors disrupted development, to varying degrees, and induced stalk cell formation in submerged culture. The inhibitors displayed the same rank order of pharmacological efficacy for stalk cell induction as they did for Rh123 transport inhibition. We also found that cerulenin, a specific inhibitor of DIF-1 biosynthesis (R. R. Kay, 1998, J. Biol. Chem. 273, 2669-2675), abolished the induction of stalk cells by each of the RhT inhibitors, and this effect could be reversed by DIF-1. Thus, DIF-1 synthesis appears to be required for the induction of stalk cells by the RhT inhibitors. Since DIF-1 is the most potent inhibitor of RhT activity, and thus a likely transport substrate itself, we propose that RhT inhibitors induce stalk cell differentiation by blocking DIF-1 export, causing DIF-1 to build up within cells. Our results provide evidence for a prespore-specific efflux pump that regulates cell fate determination, perhaps by regulating the cellular concentration of DIF-1.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Dictyostelium/cytology , Dictyostelium/metabolism , Protozoan Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/antagonists & inhibitors , Animals , Biological Transport, Active/drug effects , Cell Differentiation/drug effects , Dictyostelium/drug effects , Fluorescent Dyes/pharmacokinetics , Gene Expression Regulation, Developmental/drug effects , Genes, Protozoan , Protozoan Proteins/antagonists & inhibitors , Rhodamine 123/pharmacokinetics
6.
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
7.
Development ; 126(14): 3263-74, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10375515

ABSTRACT

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/genetics , Dictyostelium/growth & development , Gene Expression Regulation, Developmental , Protein Kinases/metabolism , Protozoan Proteins , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cyclic AMP-Dependent Protein Kinases/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Mutation , Protein Biosynthesis , Protein Kinases/genetics , Response Elements , Starvation/genetics , Starvation/metabolism
8.
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
9.
J Cell Biol ; 142(3): 735-50, 1998 Aug 10.
Article in English | MEDLINE | ID: mdl-9700162

ABSTRACT

In a search for novel members of the alpha-actinin superfamily, a Dictyostelium discoideum genomic library in yeast artificial chromosomes (YAC) was screened under low stringency conditions using the acting-binding domain of the gelation factor as probe. A new locus was identified and 8.6 kb of genomic DNA were sequenced that encompassed the whole abpD gene. The DNA sequence predicts a protein, interaptin, with a calculated molecular mass of 204,300 D that is constituted by an actin-binding domain, a central coiled-coil rod domain and a membrane-associated domain. In Northern blot analyses a cAMP-stimulated transcript of 5.8 kb is expressed at the stage when cell differentiation occurs. Monoclonal antibodies raised against bacterially expressed interaptin polypeptides recognized a 200-kD developmentally and cAMP-regulated protein and a 160-kD constitutively expressed protein in Western blots. In multicellular structures, interaptin appears to be enriched in anterior-like cells which sort to the upper and lower cups during culmination. The protein is located at the nuclear envelope and ER. In mutants deficient in interaptin development is delayed, but the morphology of the mature fruiting bodies appears normal. When starved in suspension abpD- cells form EDTA-stable aggregates, which, in contrast to wild type, dissociate. Based on its domains and location, interaptin constitutes a potential link between intracellular membrane compartments and the actin cytoskeleton.


Subject(s)
Dictyostelium/genetics , Fungal Proteins/genetics , Microfilament Proteins/genetics , Actinin/chemistry , Amino Acid Sequence , Animals , Cell Adhesion , Cell Compartmentation , Cloning, Molecular , Cyclic AMP/physiology , Dictyostelium/cytology , Fungal Proteins/chemistry , Fungal Proteins/physiology , Gene Expression Regulation, Fungal , Intracellular Membranes/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/physiology , Molecular Sequence Data , Mutagenesis , Sequence Homology, Amino Acid , Subcellular Fractions
10.
Development ; 125(12): 2291-302, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9584128

ABSTRACT

When Dictyostelium cells starve they arrest their growth and induce the expression of genes necessary for development. We have identified and characterized a protein kinase, YakA, that is essential for the proper regulation of both events. Amino acid sequence and functional similarities indicate that YakA is a homolog of Yak1p, a growth-regulating protein kinase in S. cerevisiae. Purified YakA expressed in E. coli is able to phosphorylate myelin basic protein. YakA-null cells are smaller and their cell cycle is accelerated relative to wild-type cells. When starved, YakA-null cells fail to decrease the expression of the growth-stage gene cprD, and do not induce the expression of genes required for the earliest stages of development. YakA mRNA levels increase during exponential growth and reach a maximum at the point of starvation, consistent with a role in mediating starvation responses. YakA mRNA also accumulates when cells are grown in medium conditioned by cells grown to high density, suggesting that yakA expression is under the control of an extracellular signal that accumulates during growth. Expression of yakA from a conditional promoter causes cell-cycle arrest in nutrient-rich medium and promotes developmental events, such as the expression of genes required for cAMP signaling. YakA appears to regulate the transition from growth to development in Dictyostelium.


Subject(s)
Dictyostelium/enzymology , Dictyostelium/growth & development , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , Cell Cycle , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Dictyostelium/genetics , Gene Expression Regulation , Genes, Protozoan , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Sequence Alignment , Signal Transduction
11.
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
12.
Science ; 277(5323): 251-4, 1997 Jul 11.
Article in English | MEDLINE | ID: mdl-9211856

ABSTRACT

Adenosine 3',5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) are regulators of development in many organisms. Dictyostelium uses cAMP as an extracellular chemoattractant and as an intracellular signal for differentiation. Cells that are mutant in adenylyl cyclase do not develop. Moderate expression of the catalytic subunit of PKA in adenylyl cyclase-null cells led to near-normal development without detectable accumulation of cAMP. These results suggest that all intracellular cAMP signaling is effected through PKA and that signals other than extracellular cAMP coordinate morphogenesis in Dictyostelium.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Dictyostelium/growth & development , Adenylyl Cyclases/metabolism , Animals , Cloning, Molecular , Dictyostelium/genetics , Dictyostelium/metabolism , Enzyme Activation , Gene Expression Regulation , Genes, Protozoan , Morphogenesis , Signal Transduction , Transformation, Genetic
13.
Dev Biol ; 175(2): 218-26, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8626027

ABSTRACT

When a small number of fluorescently labeled myosin II mutant cells (mhcA-) are mixed with wild-type cells and development of the chimeras is observed by confocal microscopy, the mutant cells are localized to the edges of aggregation streams and mounds. Moreover, the mutant cells stick to wild-type cells and become distorted (Shelden and Knecht, 1995). Two independent adhesion mechanisms, Contact Sites A and Contact Sites B, function during the aggregation stage and either one or both might be responsible for excluding the myosin II null cells. We have mixed mhcA- cells with cells in which the appearance of Contact Sites B is delayed (strain TL72) as well as cells which lack Contact Sites A (strain GT10) and double mutants in which both adhesion mechanisms are affected (strain TL73). In all chimeras, the mhcA- cells were distorted by interactions with the adhesion mutant cells, indicating that it does not require significant adhesive interaction to distort the flaccid cortex of mhcA- cells mhcA- cells were excluded from streams composed of cells lacking either Contact Sites A or Contact Sites B but mixed randomly with cells lacking both adhesion systems. By 10 hr of development, cells of strain TL73 acquire Contact Sites B adhesion. If cells of this strain were mixed with labeled mhcA- cells, allowed to develop for 9 hr, and then dissociated before replating, the myosin II null cells were seen to be distorted and excluded from the reaggregates. Thus, the exclusion of mhcA- cells from streams can be accomplished by either Contact Sites A or B. When chimeras of labeled TL73 and wild-type cells were made, the TL73 cells were found to be randomly mixed into aggregation streams. This result indicates that adhesive sorting does not function during aggregation and so cannot account for the exclusion of mhcA- cells from streams. We hypothesize that the flaccid cortex of mhcA- cells cannot generate sufficient protrusive force to break the contacts between adhered cells in aggregation streams but can enter streams where the cells are weakly adherent.


Subject(s)
Cell Adhesion Molecules/genetics , Dictyostelium/genetics , Fungal Proteins/physiology , Myosins/physiology , Protozoan Proteins , Amino Acid Sequence , Animals , Cell Adhesion , Cell Movement , Chimera , Dictyostelium/physiology , Fungal Proteins/genetics , Genes, Fungal , Microscopy, Confocal , Molecular Sequence Data , Morphogenesis , Myosins/deficiency , Myosins/genetics , Sequence Alignment , Sequence Homology, Amino Acid
14.
Proc Natl Acad Sci U S A ; 93(11): 5562-6, 1996 May 28.
Article in English | MEDLINE | ID: mdl-8643615

ABSTRACT

High resolution gene maps of the six chromosomes of Dictyostelium discoideum have been generated by a combination of physical mapping techniques. A set of yeast artificial chromosome clones has been ordered into overlapping arrays that cover >98% of the 34-magabase pair genome. Clones were grouped and ordered according to the genes they carried, as determined by hybridization analyses with DNA fragments from several hundred genes. Congruence of the gene order within each arrangement of clones with the gene order determined from whole genome restriction site mapping indicates that a high degree of confidence can be placed on the clone map. This clone-based description of the Dictyostelium chromosomes should be useful for the physical mapping and subcloning of new genes and should facilitate more detailed analyses of this genome. cost of silicon-based construction and in the efficient sample handling afforded by component integration.


Subject(s)
Chromosome Mapping , Chromosomes, Artificial, Yeast , Dictyostelium/genetics , Genome, Fungal , Genome, Protozoan , Animals , Base Composition , Polymorphism, Restriction Fragment Length , Reproducibility of Results , Restriction Mapping
15.
Experientia ; 51(12): 1116-23, 1995 Dec 18.
Article in English | MEDLINE | ID: mdl-8536799

ABSTRACT

Over the past ten years, powerful molecular genetic techniques have been developed to analyze gene function in Dictyostelium. DNA-mediated transformation using a variety of selections and vectors has allowed the introduction of wild-type or modified genes that are under various forms of transcriptional control. Homologous recombination is efficient and can be used to modify the genome in precise ways. In addition, it is now possible to clone genes based on their mutant phenotype alone, either by insertional mutagenesis, or by screening antisense expression cDNA libraries. Finally, a nearly complete physical map of the genome is available and so genes are easily mapped by physical techniques. We discuss many of these advances within the context of major research problems presently under study.


Subject(s)
Dictyostelium/genetics , Animals , Chromosome Mapping , Gene Expression , Gene Targeting , Mutagenesis, Insertional , Protein Engineering , RNA, Antisense/pharmacology
16.
Genetics ; 141(1): 147-57, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8536963

ABSTRACT

Detailed maps of the six chromosomes that carry the genes of Dictyostelium discoideum were constructed by correlating physically mapped regions with parasexually determined linkage groups. Chromosomally assigned regions were ordered and positioned by the pattern of altered fragment sizes seen in a set of restriction enzyme mediated integration-restriction fragment length polymorphism (REMI-RFLP) strains each harboring an inserted plasmid that carries sites recognized by NotI, SstI, SmaI, BglI and ApaI. These restriction enzymes were used to digest high molecular weight DNA prepared from more than 100 REMI-RFLP strains and the resulting fragments were separated and sized by pulsed-field gels. More than 150 gene probes were hybridized to blots of these gels and used to map the insertion sites relative to flanking restriction sites. In this way, we have been able to restriction map the 35 mb genome as well as determine the map position of more than 150 genes to with approximately 40 kb resolution. These maps provide a framework for subsequent refinement.


Subject(s)
Chromosomes, Fungal , Dictyostelium/genetics , Animals , Chromosome Mapping , Cloning, Molecular , Genes, Fungal , Polymorphism, Restriction Fragment Length
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.
Genetics ; 138(3): 665-74, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7851764

ABSTRACT

A set of 147 Dictyostelium discoideum strains was constructed by random integration of a vector containing rare restriction sites. The strains were generated by transformation using restriction enzyme-mediated integration (REMI) which results in the integration of linear DNA fragments into randomly distributed genomic restriction sites. Restriction fragment length polymorphism (RFLP) was generated in a single genomic site in each strain. These REMI-RFLP strains were used to confirm gene linkages previously supported by two other physical mapping techniques: yeast artificial chromosome (YAC) contig construction, and megabase-scale restriction mapping. New linkages were uncovered when two or more hybridization probes identified the same RFLP fragments. Probes for 100 genes have marked 53% of the RFLPs, representing greater than 22 Mb of the 40 Mb Dictyostelium genome. Alignment of these and other large fragments along each chromosome should lead to a complete physical map of the Dictyostelium genome.


Subject(s)
Dictyostelium/genetics , Genome, Fungal , Animals , Chromosomes, Artificial, Yeast , DNA Restriction Enzymes , Multigene Family , Polymorphism, Restriction Fragment Length , Restriction Mapping
20.
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
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