The TTGA-binding factor is a transcriptional regulator activated by DIF, the chlorinated hexaphenone that induces prestalk cell differentiation in Dictyostelium. The same activity also functions as a repressor, controlling stalk cell differentiation. We show that the TTGA-binding factor is a STAT protein. Like the metazoan STATs, it functions via the reciprocal interaction of a phosphotyrosine residue on one molecule with an SH2 domain on a dimerizing partner. Furthermore, it will bind specifically to a mammalian interferon-stimulated response element. In Saccharomyces cerevisiae, where the entire genomic sequence is known, SH2 domains have not been identified. It would seem, therefore, that SH2 signaling pathways arose very early in the evolution of multicellular organisms, perhaps to facilitate intercellular comunication.

No abstract

The structure of micellar aggregates formed from ionic statistical copolymers of N-acryloyl-amino acids and N-dodecylmethacrylamide in 0.05 M aqueous NaCl was studied by light scattering and fluorescence. The experimental results indicated that the tendency of the interchain aggregation increased with the hydrophobic monomer content for each series of the copolymers but decreased with hydrophobicity of the amino acid residue in the copolymers. On the other hand, while the micellar structure of the statistical copolymers strongly depended on the kind of the hydrophobe, it is little dependent on the kind of the electrolyte monomer unit at low ionic strength. Using these data, a theoretical analysis taking into account the chain stiffness effect revealed that unicore micelles formed from the ionic statistical copolymers were of flower type with a minimum loop size.

The structure and function of transcription factors of higher plants was studied by isolating cDNA clones encoding a wheat sequence-specific DNA binding protein. A hexameric nucleotide motif, ACGTCA, is located upstream from the TATA box of several plant histone genes. It has been suggested that this motif is essential for efficient transcription of the wheat histone H3 gene. A wheat nuclear protein, HBP-1 (histone DNA binding protein-1), which specifically binds to the hexameric motif, has previously been identified as a putative transcription factor. A cDNA clone encoding HBP-1 has been isolated on the basis of specific binding of HBP-1 to the hexameric motif. The deduced amino acid sequence indicates that HBP-1 contains the leucine zipper motif, which represents a characteristic property of several eukaryotic transcription factors.

T.Araki and M.Gamper contributed equally to this workDd-STAT, the protein that in part controls Dictyostelium stalk cell differentiation, is a structural and functional homolog of metazoan signal transducers and activators of transcription (STATs). Although present during growth and throughout development, Dd-STAT's tyrosine phosphorylation and nuclear localization are developmentally and spatially regulated. Prior to late aggregation, Dd-STAT is not tyrosine phosphorylated and is not selectively localized in the nucleus. During mound formation, the time at which cell-type specific gene expression initiates, Dd-STAT becomes tyrosine phosphorylated and translocates into the nuclei of all cells. The tyrosine phosphorylation and nuclear localization of Dd-STAT are induced very rapidly by extracellular cAMP through the serpentine cAMP receptor cAR1, with Dd-STAT tyrosine phosphorylation being detectable within 10 s of stimulation. This activation is independent of the only known G β subunit, suggesting that it may be G-protein independent. Nuclear enrichment of Dd-STAT is selectively maintained within the sub-population of prestalk cells that form the tip, the organizing center of the slug, but is lost in most of the other cells of the slug. This spatial patterning of Dd-STAT nuclear localization is consistent with its known role as a negative regulator of stalk-cell differentiation.

Nucleotide sequences of the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of ribosomal DNA (rDNA) from two dicot plants, carrot and broad bean, were determined. These sequences were compared with those of rice, a monocot plant, and other eukaryotic organisms. Both types of ITS region in some species of Angiospermae were the shortest among all eukaryotes so far examined and showed a wide range of variation in their G+C content, in contrast to a general trend toward very high G+C content in animals. Phylogenetic relationships of plants with animals and lower eukaryotes were considered using the nucleotide sequences of carrot and broad bean 5.8S rDNA that were determined in the present study, together with that of wheat 5.8S rRNA, which has been reported previously.

CRISPR/Cas9 has emerged in various organisms as a powerful technology for targeted gene knockout; however, no reports of editing the Dictyostelium genome efficiently using this system are available. We describe here the application of CRISPR/Cas9-mediated gene modification in Dictyostelium. The endogenous tRNA-processing system for expressing sgRNA was approximately 10 times more effective than the commonly used U6 promoter. The resulting sgRNA affected the sub-nuclear localisation of Cas9, indicating that the expression level of sgRNA was sufficiently high to form Cas9 and sgRNA complexes within the nucleus. The all-in-one vector containing Cas9 and sgRNA was transiently expressed to generate mutants in five PI3K genes. Mutation detective PCR revealed the mutagenesis frequency of the individual genes to be between 72.9% and 100%. We confirmed that all five targeting loci in the four independent clones had insertion/deletion mutations in their target sites. Thus, we show that the CRISPR/Cas9 system can be used in Dictyostelium cells to enable efficient genome editing of multiple genes. Since this system utilises transient expression of the all-in-one vector, it has the advantage that the drug resistance cassette is not integrated into the genome and simple vector construction, involving annealing two oligo-DNAs.

Nuclear protein(s) that specifically bind(s) to the upstream hexamer motif, ACGTCA, of wheat histone H3 and H4 genes has (have) been identified. Sequences homologous to this hexamer are found to be conserved in the upstream region of not only wheat histone genes but also other plant and animal histone genes. This suggests a possible role(s) for the hexamer and the nuclear protein(s) in the transcriptional regulation of the wheat histone genes. This hexamer is homologous to the upstream core sequence, TGACGTCA, which is highly conserved in some animal genes whose expression is regulated by CAMP.