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W F Loomis

Publications and source records attributed to W F Loomis.

At least 37 records · Page 2Linked to original sources
PubMed1998

Role of PKA in the timing of developmental events in Dictyostelium cells.

The cyclic AMP (cAMP)-dependent protein kinase, PKA, is dispensable for growth of Dictyostelium cells but plays a variety of crucial roles in development. The catalytic subunit of PKA is inhibited when associated with its regulatory subunit but is activated when cAMP binds to the regulatory subunit. Deletion of pkaR or overexpression of the gene encoding the catalytic subunit, pkaC, results in constitutive activity. Development is independent of cAMP in strains carrying these genetic alterations and proceeds rapidly to the formation of both spores and stalk cells. However, morphogenesis is aberrant in these mutants. In the wild type, PKA activity functions in a circuit that can spontaneously generate pulses of cAMP necessary for long-range aggregation. It is also essential for transcriptional activation of both prespore and prestalk genes during the slug stage. During culmination, PKA functions in both prespore and prestalk cells to regulate the relative timing of terminal differentiation. A positive feedback loop results in the rapid release of a signal peptide, SDF-2, when prestalk cells are exposed to low levels of SDF-2. The signal transduction pathway that mediates the response to SDF-2 in both prestalk and prespore cells involves the two-component system of DhkA and RegA. When the cAMP phosphodiesterase RegA is inhibited, cAMP accumulates and activates PKA, leading to vacuolation of stalk cells and encapsulation of spores. These studies indicate that multiple inputs regulate PKA activity to control the relative timing of differentiations in Dictyostelium.

Animals↗
PubMed1998

A cAMP-phosphodiesterase controls PKA-dependent differentiation.

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

The wacA gene of Dictyostelium discoideum is a developmentally regulated member of the MIP family.

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.

Amino Acid Sequence↗
PubMed1997

Expression of an Arabidopsis plasma membrane aquaporin in Dictyostelium results in hypoosmotic sensitivity and developmental abnormalities.

The rd28 gene of Arabidopsis thaliana encodes a water channel protein, or aquaporin, of the plasma membrane. A construct in which transcription of the rd28 cDNA is controlled by the Dictyostelium actin15 promoter was transformed into Dictyostelium discoideum cells. Transformants contained RD28 protein in their plasma membranes. When shifted to a low-osmotic-strength buffer, cells expressing rd28 swelled rapidly and burst, indicating that the plant aquaporin allowed rapid water entry in the amoebae. The rate of osmotic lysis was a function of the osmotic pressure of the buffer. We also selected transformants in which the expression of the rd28 cDNA is driven by the promoter of the prespore cotB gene. These transformants accumulated rd28 mRNA uniquely in prespore cells. In low-osmotic-strength buffer, the cotB::rd28 cells aggregated and formed normally proportioned slugs but failed to form normal fruiting bodies. The number of spores was reduced 20-fold, and the stalks of the fruiting bodies were abnormally short. The consequences of expressing RD28 in prespore cells could be partially overcome by increasing the osmolarity of the medium. Under these conditions, the cotB::rd28 cells formed fruiting bodies of more normal appearance, and the number of viable spores increased slightly. Because prespore cells have to shrink and dehydrate to form spores, it was not unexpected that expression of an aquaporin would disrupt this process, but it was surprising to find that stalk differentiation was also affected by expression of rd28 in prespore cells. It appears that osmotic stress on prespore cells alters their ability to signal terminal differentiation in prestalk cells. The results provide independent confirmation that plant aquaporins can function in the cells of other organisms, and that D. discoideum can be used to study the properties of these water channels.

Animals↗
PubMed1997

Sorting of the initial cell types in Dictyostelium is dependent on the tipA gene.

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.

Amino Acid Sequence↗
PubMed1997

Precocious sporulation and developmental lethality in yelA null mutants of Dictyostelium.

A novel developmental gene, yelA, has been found that plays as essential role in regulating terminal differentiation of Dictyostelium discoideum. Strains in which yelA is disrupted by plasmid insertion are arrested at the tight mound stage but accumulate the bright yellow pigment characteristic of mature sori. Although these mutant strains do not form fruiting bodies, many of the cells encapsulate within the mounds. Sporulation occurs about 6 hours earlier in yelA- cells than in wild-type cells, accompanied by precocious expression of a prespore gene, spiA. However, the spores are defective and lose viability over a period of several hours. Unencapsulated cells also die unless they are dissociated from the mounds and shaken in suspension. The yelA gene was isolated by plasmid rescue and found to encode a protein of 102 kDa in which the N-terminal sequence shows significant similarity to domains found in the eIF-4G subunits of the translational initiation complex eIF-4F. In wild-type cells yelA mRNA first accumulates at 8 hours of development and is maintained in both prespore and prestalk cells until culmination when it is found only is stalk cells. Mutations in yelA can partially suppress the block to sporulation in mutant strains in which either of the prestalk genes tagB or tagC is disrupted such that an encapsulation signal is not produced. It appears that premature encapsulation is normally inhibited by YelA until a signal is received from prestalk cells during culmination.

Amino Acid Sequence↗
PubMed1997

Consequences of disrupting the gene that encodes alpha-glucosidase II in the N-linked oligosaccharide biosynthesis pathway of Dictyostelium discoideum.

We have identified and disrupted the gene coding for alpha-glucosidase II in Dictyostelium discoideum. This enzyme is responsible for removing two alpha 1,3-linked glucose residues from N-linked oligosaccharides on newly synthesized glycoproteins. Mutagenesis by restriction enzyme-mediated integration (REMI) generated a clone, DG1033, which grows well but forms abnormal fruiting bodies with short, thick stalks. The strain lacks alpha-glucosidase II activity and makes incompletely processed N-linked oligosaccharides that are abnormally large and have fewer sulfate and phosphate esters. The morphological, enzymatic, and oligosaccharide profile phenotypes of the disruption mutant are all recapitulated by a targeted disruption of the normal gene. Furthermore, all of these defects are corrected in cells transformed with a normal, full-length copy of the gene. The phenotypic characteristics of DG1033 as well as chromosomal mapping of the disrupted gene indicate that it is the site of the previously characterized modA mutation. The Dictyostelium gene is highly homologous to alpha-glucosidase II genes in the human and the pig, C. elegans, and yeast. Although various cell lines have been reported to be defective in alpha-glucosidase II activity, disruption of the Dictyostelium gene gives the first example of a clear developmental phenotype associated with loss of this enzyme.

Amino Acid Sequence↗
PubMed1997

Chemotaxis to cAMP and slug migration in Dictyostelium both depend on migA, a BTB protein.

Chemotaxis in natural aggregation territories and in a chamber with an imposed gradient of cyclic AMP (cAMP) was found to be defective in a mutant strain of Dictyostelium discoideum that forms slugs unable to migrate. This strain was selected from a population of cells mutagenized by random insertion of plasmids facilitated by introduction of restriction enzyme (a method termed restriction enzyme-mediated integration). We picked this strain because it formed small misshapen fruiting bodies. After isolation of portions of the gene as regions flanking the inserted plasmid, we were able to regenerate the original genetic defect in a fresh host and show that it is responsible for the developmental defects. Transformation of this recapitulated mutant strain with a construct carrying the full-length migA gene and its upstream regulatory region rescued the defects. The sequence of the full-length gene revealed that it encodes a novel protein with a BTB domain near the N terminus that may be involved in protein-protein interactions. The migA gene is expressed at low levels in all cells during aggregation and then appears to be restricted to prestalk cells as a consequence of rapid turnover in prespore cells. Although migA- cells have a dramatically reduced chemotactic index to cAMP and an abnormal pattern of aggregation in natural waves of cAMP, they are completely normal in size, shape, and ability to translocate in the absence of any chemotactic signal. They respond behaviorally to the rapid addition of high levels of cAMP in a manner indicative of intact circuitry connecting receptor occupancy to restructuring of the cytoskeleton. Actin polymerization in response to cAMP is also normal in the mutant cells. The defects at both the aggregation and slug stage are cell autonomous. The MigA protein therefore is necessary for efficiently assessing chemical gradients, and its absence results in defective chemotaxis and slug migration.

Amino Acid Sequence↗
PubMed1997

Histidine kinases in signal transduction pathways of eukaryotes.

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.

Animals↗
PubMed1996

Developmental signal transduction pathways uncovered by genetic suppressors.

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.

Amino Acid Sequence↗
PubMed1996

A two-component histidine kinase gene that functions in Dictyostelium development.

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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome.

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.

Animals↗
PubMed1996

Cell-cell adhesion prevents mutant cells lacking myosin II from penetrating aggregation streams of Dictyostelium.

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.

Amino Acid Sequence↗
PubMed1996

Initial cell type divergence in Dictyostelium is independent of DIF-1.

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.

ATP-Binding Cassette Transporters↗
PubMed1996

The Dictyostelium dual-specificity kinase splA is essential for spore differentiation.

We have studied the structure and function of the Dictyostelium kinase splA. A truncated form of the splA protein exhibited primarily tyrosine kinase activity in vitro; however, it also autophosphorylated on serine and threonine residues. The kinase domain of splA exhibits approximately 38% identity to the CTR1 kinase of Arabidopsis, which is a member of the Raf family. Outside its kinase domain, splA shares homology with the byr2 kinase of S. pombe. By aligning the sequences of splA, byr2 and STE11, a homologue of byr2 in S. cerevisiae, we have identified a conserved motif that is also found in members of the Eph family of growth factor receptor tyrosine kinases. SplA is expressed throughout development with a peak during the mound stage of morphogenesis. Strains in which the splA gene had been disrupted completed fruiting body formation; however, spore cells spontaneously lysed before completing their differentiation. Northern analysis revealed the expression of the prespore marker cotB and the prestalk markers ecmA and ecmB in the mutant strain during development. The spore differentiation marker spiA was detected in the mutant spores both by northern and immunoblotting, but these cells failed to assemble spore coats. Immunoblot analysis of the developmental pattern of tyrosine phosphorylation revealed a protein that was phosphorylated in mutants but was not phosphorylated in the wild-type cells. SplA is a novel dual specificity kinase that regulates the differentiation of spore cells.

Amino Acid Sequence↗
PubMed1995

Consensus phylogeny of Dictyostelium.

The evolutionary relationship of Dictyostelium discoideum to the yeasts, fungi, plants, and animals is considered on the basis of physiological, morphological and molecular characteristics. Previous analyses of five proteins indicated that Dictyostelium diverged after the yeasts but before the metazoan radiation. However, analyses of the small ribosomal subunit RNA indicated divergence prior to the yeasts. We have extended the molecular phylogenetic analyses to six more proteins and find consistent evidence for a more recent common ancestor with metazoans than yeast. A consensus phylogeny generated from these new results by both distance matrix and parsimony analyses establishes Dictyostelum's place in evolution between the yeasts Saccharomyces cerevisiae and Schizzosaccharomyces pombe and the worm Caenorhabditis elegans.

Animals↗
PubMed1995

Genetic networks.

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Biological Evolution↗