Yoshimasa Tanaka

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal–fungal lineage after the plant–animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Periodic Signaling Controlled by an Oscillatory Circuit That Includes Protein Kinases ERK2 and PKA

Self-regulating systems often use robust oscillatory circuits. One such system controls the chemotactic signaling mechanism of Dictyostelium, where pulses of adenosine 3′,5′-monophosphate (cAMP) are generated with a periodicity of 7 minutes. We have observed spontaneous oscillations in activation of the mitogen-activated protein (MAP) kinase ERK2 that occur in phase with peaks of cAMP, and we show that ERK2 modulates cAMP levels through the phosphodiesterase RegA. Computer modeling and simulations of the underlying circuit faithfully account for the ability of the cells to spontaneously generate periodic pulses during specific stages of development. Similar oscillatory processes may occur in cells of many different species.

Dictyostelium transcriptional host cell response upon infection with Legionella.

Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. Investigation of a 48u2003h time course of infection revealed several clusters of co‐regulated genes, an enrichment of preferentially up‐ or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24u2003h after infection. A detailed analysis of the 24u2003h time point post infection was performed in comparison to three controls, uninfected cells and co‐incubation with Legionella hackeliae and L. pneumophilaΔdotA. One hundred and thirty‐one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host‐pathogen interactions and for further studies on the Dictyostelium response to Legionella infection.

Changing Patterns of Gene Expression in Dictyostelium Prestalk Cell Subtypes Recognized by In Situ Hybridization with Genes from Microarray Analyses

ABSTRACT We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

The mitochondrial DNA of Dictyostelium discoideum: Complete sequence, gene content and genome organization

Abstract We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564u2009bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4u2009kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.

A STAT-regulated, stress-induced signalling pathway in Dictyostelium

The Dictyostelium stalk cell inducer differentiation-inducing factor (DIF) directs tyrosine phosphorylation and nuclear accumulation of the STAT (signal transducer and activator of transcription) protein Dd-STATc. We show that hyperosmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyperosmotic stress is known to elevate intracellular cGMP and cAMP levels, and the membrane-permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, whereas 8-bromo-cAMP is a much less effective inducer. Surprisingly, however, Dd-STATc remains stress activatable in null mutants for components of the known cGMP-mediated and cAMP-mediated stress-response pathways and in a double mutant affecting both pathways. Also, Dd-STATc null cells are not abnormally sensitive to hyperosmotic stress. Microarray analysis identified two genes, gapA and rtoA, that are induced by hyperosmotic stress. Osmotic stress induction of gapA and rtoA is entirely dependent on Dd-STATc. Neither gene is inducible by DIF but both are rapidly inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a transcriptional activator in a stress-response pathway and the pharmacological evidence, at least, is consistent with cGMP acting as a second messenger.

A mutant form of the tax protein of bovine leukemia virus (BLV), with enhanced transactivation activity, increases expression and propagation of BLV in vitro but not in vivo.

ABSTRACT In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.

Construction of a gamete-enriched gene pool and RNAi-mediated functional analysis in Dictyostelium discoideum

Macrocysts in Dictyostelium discoideum possess prototypic features of sexual reproduction and are useful for understanding the basic mechanisms of the reproductive process. Here, we randomly analyzed 1,071 gamete cDNAs, and then constructed a gamete-specific subtraction library, FC-IC. Nucleotide sequences of all 903 FC-IC clones were determined and clustered into 272 independent genes. Expression analysis based on real-time RT-PCR revealed 67 gamete-enriched genes, among which those involved in signal transduction and multicellular organization are prevalent. One of them, FC-IC0003, appeared also to be mating-type specific, and was named gmsA. RNAi-mediated silencing as well as disruption of gmsA reduced the cellular competency for sexual cell fusion, indicating the involvement of this gene in the sexual development of D. discoideum.

CODON USAGE, GENETIC CODE AND PHYLOGENY OF DICTYOSTELIUM DISCOIDEUM MITOCHONDRIAL DNA AS DEDUCED FROM A 7.3-KB REGION

We have sequenced a region (7 376-bp) of the mitochondrial (mt) DNA (54 kb) of the cellular slime mold, Dictyostelium discoideum. From the DNA and amino-acid sequence comparisons with known sequences, genes for ATPase subunit 9 (ATP), cytochrome b (CYTB), NADH dehydrogenase subunits 1, 3 and 6 (ND1, ND3 and ND6), small subunit rRNA (SSU rRNA) and seven tRNAs (Arg, Asn, Cys, Lys, f-Met, Met and Pro) have been identified. The sequenced region of the mtDNA has a high average A+T-content (70.8%). The A+T-content of protein-genes (73.6%) is considerably higher than that of RNA genes (61.3%). Even with the strong AT-bias, the genetic code employed is most probably the universal one. All seven tRNAs are able to form typical clover leaf structures. The molecular phylogenetic trees of CYTB and SSU rRNA suggest that D. discoideum is closer to green plants than to animals and fungi.

Purification and some properties of Δ2-isopentenylpyrophosphate:5′AMP Δ2-isopentenyltransferase from the cellular slime mold Dictyostelium discoideum

Abstract Δ 2 -Isopentenylpyrophosphate:5′AMP Δ 2 -isopentenyltransferase, which catalyzes the formation of isopentenyl-AMP from Δ 2 -isopentenylpyrophosphate and 5′AMP, was purified 6800-fold from the fruiting body of the cellular slime mold Dictyostelium discoideum using several separation procedures including 5′AMP ox-red AH-Sepharose 4B affinity column chromatography. The final preparation was very unstable and lost its activity in a day. Various properties of the 1000-fold-purified enzyme preparation were examined. The molecular mass was 40,000 ± 2000 Da, as determined by Sephadex G-100 superfine gel filtration. The divalent metal ions Mn 2+ , Zn 2+ , and Mg 2+ profoundly affected the enzymatic activity depending on their concentration, and also altered the optimum pH and temperature. Of the compounds tested, 5′AMP was the best acceptor of the isopentenyl group and, interestingly, ADP also served as a substrate, being 60–80% as effective as 5′AMP. Adenine, adenosine, and ATP were not substrates for this enzyme. Under the optimum assay conditions (pH 7.0, 1 m m Zn 2+ , and 25 °C) the K m values for 5′AMP and Δ 2 -isopentenylpyrophosphate were 1.0 × 10 −7 m and 2.2 × 10 −6 m , respectively.