Search PubMed⌕ Search

Biomedical subjects

W F Loomis

Publications and source records attributed to W F Loomis.

At least 55 records · Page 3Linked to original sources
PubMed1995

Mitochondrial DNA replication but no nuclear DNA replication during development of Dictyostelium.

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.

Animals↗
PubMed1995

A multidrug resistance transporter/serine protease gene is required for prestalk specialization in Dictyostelium.

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.

ATP-Binding Cassette Transporters↗
PubMed1995

Whole-mount in situ hybridization of cell-type-specific mRNAs in Dictyostelium.

We have been able to hybridize nonradioactive probes from cell-type-specific genes to fixed whole-mounts prepared at the mound, slug, and culminant stages of Dictyostelium development. The cellular patterns of labeling with probes from the prespore gene, cotB, and the prestalk genes, ecmA and ecmB, confirmed the patterns seen in strains carrying reporter constructs in which the regulatory regions of these genes drive beta-galactosidase. This technique permits the direct observation of protein synthetic capacity from characterized genes without the need of generating transformed lines carrying specific reporter constructs. Moreover, the pattern is not complicated by a previous developmental history of gene expression.

Animals↗
PubMed1995

Protein kinase A is a positive regulator of spore coat gene transcription in Dictyostelium.

The cotA, cotB, and cotC genes encode the major spore coat proteins of Dictyostelium. All three cot genes are coordinately expressed as aggregation is nearing completion. Induction and maintenance of their expression is dependent upon the presence of extracellular cAMP. We show that expression of a dominant inhibitor of the cAMP dependent protein kinase (PKA) in prespore cells greatly reduces the transcription rates of the cotB and cotC genes. All three cot genes contain, in their upstream regulatory regions, short sequence elements that have a high content of cytosine and adenosine residues. These CA-rich sequences are essential for optimal cot gene transcription. We show that expression of the dominant PKA inhibitor results in a greatly reduced level of the binding activity that recognizes the CA-rich sequences upstream of the cotB gene. Thus PKA acts, either directly or indirectly, to control expression of the cot genes and it may do so by modulating the activity of a DNA binding protein. However, we find that mutant cells where PKA is constitutively active still require exogenous cAMP for optimal cot gene expression in dissociated cells, suggesting that a separate, PKA-independent, signalling pathway is also involved in the regulation of cot gene expression by extracellular cAMP.

Animals↗
PubMed1995

A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium.

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.

Adenylyl Cyclases↗
PubMed1995

Integrated maps of the chromosomes in Dictyostelium discoideum.

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.

Animals↗
PubMed1994

Discovery of myosin genes by physical mapping in Dictyostelium.

The diversity of the myosin family in a single organism, Dictyostelium discoideum, has been investigated by a strategy devised to rapidly identify and clone additional members of a gene family. An ordered array of yeast artificial chromosome clones that encompasses the Dictyostelium genome was probed at low stringency with conserved regions of the myosin motor domain to identify all possible myosin loci. The previously identified myosin loci (mchA, myoA-E) were detected by hybridization to the probes, as well as an additional seven previously unidentified loci (referred to as myoF-L). Clones corresponding to four of these additional loci (myoF, myoH-J) were obtained by using the isolated yeast artificial chromosomes as templates in a PCR employing degenerate primers specific for conserved regions of the myosin head. Sequence analysis and physical mapping of these clones confirm that these PCR products are derived from four previously unidentified myosin genes. Preliminary analysis of these sequences suggests that at least one of the genes (myoJ) encodes a member of a potentially different class of myosins. With the development of whole genome libraries for a variety of organisms, this approach can be used to rapidly explore the diversity of this and other gene families in a number of systems.

Amino Acid Sequence↗
PubMed1994

LagC is required for cell-cell interactions that are essential for cell-type differentiation in Dictyostelium.

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.

Actins↗
PubMed1994

Structural roles of the spore coat proteins in Dictyostelium discoideum.

The integrity of spores formed by mutant strains of Dictyostelium discoideum lacking the major spore coat proteins, SP96, SP70, or SP60, was compared to that of wild-type strains. Single, double, and triple knock-out strains developed normally and produced spores which were indistinguishable from wild-type spores by light or electron microscopy. However, the mutant strains were susceptable to staining with the lectin, ricin A, which recognizes a galactose-rich polysaccharide that is normally hidden by overlying spore coat proteins. The intensity of staining with fluorescently labeled ricinA increased as the spore coat proteins were incrementally lost. While these results indicate that the major outer spore coat proteins are not essential for the construction of a multi-layered spore coat in Dictyostelium, they show that the spores are more porous which might make them at risk to predators before germination.

Animals↗
PubMed1994

CRAC, a cytosolic protein containing a pleckstrin homology domain, is required for receptor and G protein-mediated activation of adenylyl cyclase in Dictyostelium.

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.

Adenylyl Cyclases↗
PubMed1994

REMI-RFLP mapping in the Dictyostelium genome.

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.

Animals↗
PubMed1994

Progression of an inductive signal activates sporulation in Dictyostelium discoideum.

spiA, a marker for sporulation, is expressed during the culmination stage of Dictyostelium development, when the mass of prespore cells has moved partly up the newly formed stalk. Strains containing a full-length spiA promoter/lacZ fusion were stained for beta-galactosidase activity at intervals during development. The results indicate that expression of spiA initiates in prespore cells at the prestalk/prespore boundary (near the apex) and extends downward into the prespore mass as culmination continues. A spatial gradient of staining expands from the top of the prespore mass and intensifies until the front of activation reaches the bottom, whereupon the entire region stains darkly. The spiA promoter can be deleted to within 301 bp of the transcriptional start site with no effect on the relative strength, timing or spatial localization of expression. Further 5' deletions from -301 to -175 reduce promoter strength incrementally, although timing and spatial expression are not affected. Deletions to -159 and beyond result in inactive promoters. Treatment of early developmental structures with 8-Br-cAMP in situ activates the intracellular cAMP-dependent protein kinase (PKA) and precociously induces spiA expression and sporulation. The absence of an apparent gradient of staining in these structures suggest that PKA is equivalently activatable throughout the prespore region and that all prespore cells are competent to express spiA. Thus, we postulate that the pattern of expression of spiA reveals the progression of an inductive signal for sporulation and suggest that this signal may originate from the prestalk cells at the apex.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Enhancer regions responsible for temporal and cell-type-specific expression of a spore coat gene in Dictyostelium.

The extracellular spore coat of Dictyostelium discoideum is composed of three major proteins, SP96, SP70, and SP60, encoded by the cotA, cotB, and cotC genes, respectively. The spore coat proteins are coordinately synthesized in prespore cells shortly after aggregation, stored in prespore vesicles during the slug stage, and secreted during encapsulation of spores. We have ligated various portions of the upstream region of cotB to lacZ such that a protein consisting of the first nine amino acids of SP70 fused to beta-galactosidase is synthesized in prespore cells. Individual cells that accumulate the enzyme can be observed in situ during early aggregation due to the sensitivity of the assay. We have found that prespore cells first appear in a random distribution throughout the aggregates with no indication of spatial localization. They subsequently sort out from prestalk cells that form a tip on the aggregates. The cotB regulatory region was subdivided into a proximal and a distal region, each of which could independently direct proper temporal and cell-type control. Transcriptional activity directed by these two regions appears to be additive in the full-length regulatory region. The proximal region was shown to be complex in that removal of certain portions partially reduced transcriptional activity while removal of other portions abolished all activity. Nevertheless, cells transformed with constructs showing attenuated activity expressed the fusion gene at the proper time in development and the activity was localized to prespore cells. The cis-acting regions responsible for all aspects of cotB regulation appear to be closely opposed within the minimal essential sequence of the proximal region.

Animals↗
PubMed1993

Cell type regulation in response to expression of ricin A in Dictyostelium.

Expression of ricin A in either prespore or prestalk cells of Dictyostelium discoideum results in cell-autonomous lethality. Strains expressing the toxic gene under the control of a prestalk-specific regulatory region fail to culminate or form stalks, but form spores normally. Strains expressing ricin A under the control of a prespore-specific regulatory region form neither spores nor stalks. Regulation of the cell types results in conversion of prestalk cells to prespore cells when the prespore cells are poisoned. The newly converted cells then express ricin A and die. In contrast, we could not detect any significant conversion of prespore cells to prestalk cells when the prestalk cells are poisoned under our experimental conditions. This regulation of cell types suggests that the tendency of prestalk cells to regulate and become prespore cells is inhibited by the already established prespore cells. It appears that prespore cells control prestalk cell regulation by producing an inhibitor of prespore differentiation to which they themselves are insensitive.

Animals↗
PubMed1992

Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA.

Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants. We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid DNA alone is introduced. Restriction enzyme-mediated integration generates insertions into genomic restriction sites in an apparently random manner, some of which cause mutations. About 1 in 400 of the Dictyostelium transformants displayed arrested or aberrant development. The integrated plasmid, along with flanking genomic DNA, was excised from some of these mutants, cloned in Escherichia coli, and used to transform other Dictyostelium cells. Homologous recombination within the flanking sequences resulted in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes. This method of insertional mutagenesis should be useful for tagging, and subsequent cloning, of many developmentally important genes that can be identified by their mutant phenotypes.

Animals↗
PubMed1992

Physical mapping of genes to specific chromosomes in Dictyostelium discoideum.

Cloned genes were used to probe a highly redundant library of large cloned fragments of the Dictyostelium discoideum genome carried in yeast artificial chromosomes (YACs). Each gene recognized several independent YAC clones, thereby grouping them into a contig. Individual YACs were arranged within the contig by positioning genes relative to rare restriction sites and the YAC ends. Genes that had been previously assigned to one of the six linkage groups by parasexual genetics were used to establish physically mapped regions on specific chromosomes. Previously unmapped genes were assigned to specific chromosomes when they recognized members of a mapped contig. Linkage was confirmed by congruence of large-scale restriction maps centered on either the previously mapped or the newly mapped genes. At present, the chromosome-assigned map segments comprise approximately 50% of the genome. About half of each map segment is covered by overlapping YACs.

Animals↗