Developmentally regulated protein-tyrosine kinase genes in Dictyostelium discoideum.

Dictyostelium discoideum, an organism that undergoes development and that is amenable to biochemical and molecular genetic approaches, is an attractive model organism with which to study the role of tyrosine phosphorylation in cell-cell communication. We report the presence of protein-tyrosine kinase genes in D. discoideum. Screening of a Dictyostelium cDNA expression library with an anti-phosphotyrosine antibody identifies fusion proteins that exhibit protein-tyrosine kinase activity. Two distinct cDNAs were identified and isolated. Though highly homologous to protein kinases in general, these kinases do not exhibit many of the hallmarks of protein-tyrosine kinases of higher eucaryotes. In addition, these genes are developmentally regulated, which suggests a role for tyrosine phosphorylation in controlling Dictyostelium development.     

Phototaxis genes on linkage group V in Dictyostelium discoideum

Abstract During the course of mapping and complementation analysis of phototaxis ( pho ) mutations in Dictyostelium discoideum we have assigned to linkage group V three mutant pho alleles belonging to complementation groups phoG and phoK . These are the first genetic markers with an easily recognizable phenotype to be found on this linkage group.     

Chromosomal mapping of tRNA genes from Dictyostelium discoideum

Summary Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA^Val(GUU) and a tRNA^Val(GUA) gene. The method used involved a combination of classitics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA^Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F,), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VIII (A, H). Six tRNA^Val(GUA) genes from the NC4 isolate were mapped; one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).     

Multiple genes for cell surface cAMP receptors in Dictyostelium discoideum

We have cloned and characterized three genes (CAR1, CAR2, CAR3) encoding potential cell surface, cyclic adenosine 3':5' monophosphate (AMP) receptors from Dictyostelium discoideum. The three proteins are predicted to be substantially similar in amino acid sequence throughout most of their transmembrane (TM) and loop domains but are distinctly different in their carboxyl terminal segments. In addition, all three genes possess an intron which interrupts an equivalent codon of TM3. CAR1 is expressed early in development when the cAMP relay system is being established. As development proceeds multiple size forms of CAR1 RNA are detected which apparently result from differences in their 5'-untranslated regions. Late in development levels of CAR1 RNA decrease. In contrast, CAR2 encodes a single sized RNA which is expressed only during postaggregative development. CAR3 expression is approximately 10% of CAR1 during early development, is maximal during tight aggregate formation but declines thereafter. Only one size class of CAR3 mRNA is detected throughout development. Because RNA for each of the three genes is present in postaggregative cells, it was of interest to determine the cell type distribution of each RNA. Gene-specific probes were hybridized to RNAs isolated from cells of Percoll gradient-enriched prespore and prestalk fractions and relative levels of hybridization compared. CAR1 and CAR3 show approximately the same pattern of accumulation; a 3-4 fold enrichment in prestalk cells. CAR2, however, is highly enriched in prestalk cells, more than 10 fold relative to prespore cells.     

Identification and characterization of two flavohemoglobin genes in Dictyostelium discoideum

Flavohemoglobins are being identified in an expanding number of prokaryotes and unicellular eukaryotes. These molecules consist of an N-terminal hemoglobin domain and a C-terminal oxidoreductase domain, and are considered to function in storage or as sensors for O2, and in defense against oxidative stress and/or NO. However, their physiological significance has not yet been determined. Here, we isolated and analyzed two flavohemoglobin genes of Dictyostelium discoideum, DdFHa and DdFHb, which lie close to each other in the genome. DdFHs were induced by submerged conditions, and enriched in the sexually mature cells of D. discoideum. Although they were not essential for growth or development under standard laboratory conditions, disruption of both genes caused an increase in number of large but uninuclear cells, and hypersensitivity to higher concentrations of glucose and to NO releasers. These results indicate that DdFHs are responsible for transducing NO signals to maintain normal cellular conditions against environmental stresses.     

Aggregation in Dictyostelium discoideum

Cultured peritoneal macrophages have previously been shown to release a potent mitogen for mesenchymal cells. Peritoneal macrophages are derived from peripheral blood monocytes, one of the principal inflammatory cells associated with numerous tissue responses to injury. Cultured human monocytes can be activated by endotoxin or concanavalin A to secrete a potent growth factor(s) that is active on human smooth muscle cells, human fibroblasts and 3T3 cells. The optimal conditions for activation of monocyte release of this monocyte-derived growth factor(s) (MDGF) were to expose 5-day-old monocyte cultures (initially plated at 6.8 × 10⁵ cells/ml medium) to 10 μg/ml endotoxin or 6 μg/ml concanavalin A for approximately 20 hr. Monocytes can secrete MDGF into serum-free medium supplemented with 0.15% bovine serum albumin. MDGF stimulates both DNA synthesis and increase in cell number and is trypsin-sensitive, heat labile and nondialyzable. The relationship of MDGF to other monocyte products and its potential importance in wound repair and atherogenesis are discussed.     

Chemiluminescence in Dictyostelium discoideum

Abstract Oxygen radicals generated during oxidative metabolism participate in chemical reactions resulting in light emission. Chemiluminescence is used therefore to measure their production. We have shown that starvation and heat shock induce chemiluminescence in Dictyostelium discoideum . The peak light emission was found to occur about 4 h after the onset of starvation. The optimum temperature for chemiluminescence by starving amoebae was about 33°C. The heat shock inducibility of chemiluminescence was maximal at the beginning of development. Our results are consistent with suggestions that the product(s) of perturbed mitochondrial metabolism might be intracellular signal(s) controlling gene expression in stressed cells. They also suggest a role for intracellular stress signal(s) in the initiation of development in Dictyostelium by starvation.     

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.     

Two cyclic AMP-regulated genes from Dictyostelium discoideum encode homologous proteins

Expression of the 7E and 2C genes late in Dictyostelium development ceases upon cell disaggregation but, in contrast to many other genes we have studied, expression is fully restored by exogenous cAMP (A. J. Richards et al., submitted). The 7E and 2C genes encode polypeptides of similar size (9220 and 10573 Daltons, respectively), each of which contains an unusually high proportion of serine plus glycine residues (41% and 59%, respectively). Each protein possesses a relatively serine-rich N-terminus and glycine-rich C-terminus and contains the conserved sequence S(X)SSS(X2)SS(X)SS(X2)SFGS. These data suggest that genes 7E and 2C may have arisen by duplication of a common ancestor. Computer analysis indicates that both gene products are probably intracellular structural proteins that form extended coil structures.     

Ribosomal proteins are encoded by single copy genes in Dictyostelium discoideum

Five recombinant plasmids which encode ribosomal proteins (r-proteins) from Dictyostelium discoideum have been isolated. Poly(A) + RNA was size-fractionated by preparative agarose gel electrophoresis and a fraction encoding proteins of less than 35 kDa was used to construct a cDNA library in the plasmid vector pBR322. Individual clones from the library were screened by hybrid-selected translation and those encoding r-proteins were identified by co-migration of the translation products in two-dimensional gel electrophoresis with marker proteins purified from Dictyostelium ribosomes. Initial characterization using the five cDNA plasmids indicates that these r-proteins are encoded by single copy genes and that they are not tightly clustered in the genome.     

Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.     

Prespore-inducing factors in Dictyostelium discoideum

Abstract. In an attempt to investigate the pathway of prespore differentiation in the cellular slime mold, Dictyostelium discoideum , we examined the requirements for prespore differentiation under conditions involving no cellular interactions. Aggregation-competent cells of the wild-type NC4 strain incubated in an isolated state were found to differentiate into prespore cells depending on the presence of cAMP, bovine serum albumin, and the prespore inducing factors (PIF) obtained from the conditioned media. Vegetative cells did not differentiate into prespore cells under these conditions. The prespore-inducing activity found in extracts of cells was developmentally regulated and induced by cAMP. PIF was of a low molecular mass (150–400 kilodaltons), was heat, alkali, and acid stable, and was extractable from an acidified water phase using ethylacetate. Purification of the PIF in ethylacetate extracts by gel-permeation chromatography followed by reverse-phase chromatography revealed two major peaks with prespore-inducing activity. On mixing these two fractions, the original activity was restored, indicating that PIF is composed of at least two components.     

Accumulation of alpha-mannosidase-1 in Dictyostelium discoideum requires many developmentally essential genes

alpha-Mannosidase-1, one of the earliest known developmentally controlled gene products in the cellular slime mold Dictyostelium discoideum, accumulates intracellularly during both axenic growth and development. The accumulation of alpha-mannosidase-1 activity prematurely ceases in all of 125 randomly isolated aggregation-deficient mutants at discrete times in development resulting in significantly reduced levels of cellular enzyme activity. This suggests that, unlike other developmentally controlled enzymes in this organism, the continued accumulation of alpha-mannosidase-1 activity is controlled by a large number of genes essential for early development. alpha-Mannosidase-1 misregulation and the aggregation-deficient phenotype are caused by the same mutation since (1) morphological revertants exhibit a coreversion to both fruiting ability and wild-type alpha-mannosidase-1 accumulation and (2) normal enzyme accumulation depends on the ability to aggregate and ultimately fruit in a conditional aggregation-deficient mutant. This type of regulation does not appear to be due to differences in enzyme secretion or changes in the overall rate of total protein synthesis. Aggregation-deficient mutants continue to synthesize protein beyond the time in development at which alpha-mannosidase-1 accumulation ceases. Our studies indicate that most of the 50-125 genes required for aggregation in Dictyostelium are also required for the normal accumulation of alpha-mannosidase-1 activity.     

Developmental decisions in Dictyostelium discoideum

Dictyostelium discoideum is an excellent system in which to study developmental decisions. Synchronous development is triggered by starvation and rapidly generates a limited number of cell types. Genetic and image analyses have revealed the elegant intricacies associated with this simple development system. Key signaling pathways identified as regulating cell fate decisions are likely to be conserved with metazoa and are providing insight into differentiation decisions under circumstances where considerable cell movement takes place during development.