Masao Tasaka

Molecular cloning of cell-type-specific cDNAs exhibiting new types of developmental regulation in Dictyostelium discoideum

By screening cDNA libraries from NC-4 cells, we have obtained five prespore-specific and two prestalk-specific cDNA clones. The two classes of prespore genes began to be expressed at the tipped aggregate stage: one class was expressed throughout development, while the other was shut off during culmination. Another class of prespore genes was expressed in vegetative cells but only in prespore cells at the later stages. Prestalk-specific genes were first expressed at the tipped aggregate stage concurrently with the two prespore gene classes, indicating the importance of this stage for transcriptional regulation in both prestalk and prespore differentiation. Except for the first prespore gene class, these represent new cell-type-specific genes as to their temporal pattern of expression.

Isolation and characterization of spore coat protein (sp96) gene of Dictyostelium discoideum

A cDNA library was constructed from poly(A)+ RNA isolated from slug cells of Dictyostelium discoideum, using lambda gt11 phage, and screened with an antiserum specific for the spore coat protein sp96. A positive clone was obtained and the gene product was identified as sp96. The sp96 mRNA is 2.2 kb in size, and it starts to accumulate at the tipped aggregate stage only in prespore cells. Southern analysis using nuclear DNA established that the sp96 gene is unique. Two genomic clones containing the sp96 gene were isolated and the sequence of the gene established. The coding region contains a long open reading frame interrupted by a single intron.

cAMP regulation of the expression of prespore-specific genes, SP96 and Dp87, in disaggregated slug cells of Dictyostelium discoideum

By Northern transfer and in vitro transcription assays, we examined how cAMP controlled the expression of prespore genes, Dp87 and SP96, in disaggregated slug cells of Dictyostelium discoideum. The transcription of these genes was halted soon after disaggregation and the remaining mRNAs were completely lost within 2 h. Addition of cAMP to cells which had lost the mRNAs induced the transcription of these genes, and the mRNAs were re-accumulated after a lag period of 30 min. The cAMP signal was transduced through the cell surface receptor. Protein synthesis was not needed for the induction of the transcription but was required for the accumulation of the mRNAs. We conclude that prespore gene expression is controlled by cAMP in two different ways: direct induction of transcription of the genes, and stabilization of the transcribed mRNAs by a protein(s) synthesized after addition of cAMP.

Transcriptional Regulation of Cell-Type-Enriched Genes during Development in Dictyostelium discoideum Analyzed by Nuclear Run-On Assays

We have previously isolated several cell‐type‐enriched mRNAs of Dictyostelium discoideum. Since the temporal pattern of appearance and cell‐type‐enrichment of these RNAs were examined only by determining their accumulation, it was unclear whether their accumulation is regulated at the transcription level or the post‐transcriptional level. To distinguish between these two possibilities, we examined the temporal and cell‐type‐enriched transcription of several of these genes by nuclear run‐on assay. The results suggest that some genes are controlled in both temporal accumulation and cell‐type‐enrichment at the transcriptional level, but post‐transcriptional regulation is also important for regulating cell‐type enrichment in the case of some other genes.

Expression of an altered cAMP binding protein by rapid-developing strains of Dictyostelium discoideum

Immunoblotting with a monoclonal antibody raised against a novel cAMP binding protein termed CABP1 revealed that the molecular weights of the two CABP1 subunits are altered in certain strains of Dictyostelium discoideum. Cell-free translation followed by immunoprecipitation showed that the altered CABP1 polypeptides are derived from primary translation products. In addition, the affinity of the altered CABP1 for cAMP is much higher than the wild-type form. Morphologically, these strains are indistinguishable from other wild-type strains except that their developmental phase is considerably shorter. The rapid developers also exhibit a precocious appearance of CABP1. These results indicate a good correlation between an altered CABP1 and rapid development.

Regulation of Prestalk‐specific Acid Phosphatase in Dictyostelium discoideum

Acid phosphatase–2, as characterized by gel electrophoresis under non‐denaturing conditions, is a convenient marker for prestalk cells in Dictyostelium discoideum. We have purified this prestalk‐specific enzyme and have examined its regulation during development. Under denaturing conditions, the enzyme has a molecular weight of 50,000 and an isoelectric point of 4.0. On the other hand, acid phosphatase‐I have a Mr‐55,000 polypeptide (AP1–55) and a minor Mr‐50,000 polypeptide (AP1–50) and both have diffuse isoelectric point from 3.4 to 4.1. Using monoclonal antibodies directed against acid phosphatase‐2 as probes, we showed that some acid phosphatase‐2 are newly synthesized at slug stage and some are converted from AP1–50 which was synthesized during ealy development.