A cDNA clone SSJ337 (accession no. AF161253) of 1230 bp, encoding a catalytic subunit of protein phosphatase 4, was selected as one of the clones expressed specifically in prestalk cells from a cDNA library of D. discoideum slugs. Cells transformed with a knockout construct of SSJ337 showed an aberrant and tiny fruiting-body formation with a short stalk. A knockout mutant, SSJ337KO was allowed to develop much slower than a wild-type AX2 after the post-aggregation stage. This suggested that the SSJ337 cDNA clone has played an important role especially in the later development of Dictyostelium discoideum. Results from Northern blotting, analysis showed that transcripts for SSJ337 were accumulated at 16 h to 24 h after starvation began.
Dictyostelium discoideum is a unique organism having a single amoeba stage and a multicellular stage in its life cycle. D. discoideum has been used as a model organism to study development or differentiation mechanisms. After starvation, D. discoideum cells aggregate at 8 h and eventually form a fruiting body consisting of spore and stalk cells at 24 h [
The knockout mutants for genes involved in the development of D. discoideum show aberrant development. A cAMP receptor subtype 2, CAR2, is required for the cAMP-directed sorting of prestalk cells during pattern formation of an aggregation mound, and the knockout mutant cells remain in the tight aggregation stage [
Wild-type AX2 was cultivated at 22˚C in HL5 medium. A knockout mutant for a SSJ337 cDNA clone, SSJ337KO, was grown in HL5 medium containing 10 μg/ml Blasticidin S (Funakoshi, Tokyo, Japan). Development was begun by washing the cells in 17 mM phosphate buffer, pH 6.1. The cells were suspended in the same buffer at a density of 1 × 107/ml. They were agitated on a rotary shaker at 150 rpm, or spread on agar plates, and incubated at 22˚C for the indicated times as reported previously [
A knockout construct for the SSJ337 cDNA clone (accession no. AF161253), which was primarily sequenced by us as a clone of the “Dictyostelium cDNA project in Japan, http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html”, was prepared as follows (
A cDNA probe was labeled with digoxigenin (DIG) by the random hexamer procedure using a DIG DNA labeling kit (Roche Diagnostics, Mannheim, Germany). Genomic DNAs and total RNAs were extracted with ISOGEN (Nippon gene, Japan). Total RNAs were extracted from wild-type AX2 cells starved for 0, 8, 16 or 24 h on non-nutrient agar plates, and Northern blotting analysis was conducted using SSJ337 cDNA as a probe. Ten μg of total RNAs was separated on formaldehydeagarose gel. Five μg of genomic DNAs was digested with an appropriate restriction enzyme and separated on agarose gel. After agarose gel electrophoresis, agarose gels were transferred onto HybondTMN+nylon membrane (GM Healthcare, Buckinghamshire, UK) with VacuGeneXL (GM Healthcare). Hybridization was carried out at 50˚C in DIG easy hyb (Roche Diagnostics) for Southern blotting analysis and at 45˚C for Northern blotting analysis.
To identify novel genes involved in fruiting-body forma-
tion, we prepared knockout constructs by inserting a BSR cassette into each appropriate restriction-enzyme site of several dozens cDNA expressed specifically in prestalk cells from a Dictyostelium discoideum slug cDNA library. Of them, a knockout construct for SSJ337 cDNA clone (SSJ337-Bsr) was prepared by inserting the BSR cassette into Xba I of SSJ337 cDNA. Three knockout mutants transformed with SSJ337-Bsr (SSJ337KO) and isolated independently, showed the same aberrant and tiny fruiting bodies. In particular, the stalks of transformants were much shorter than those of wild-type AX2. Southern blotting analysis of transformants, using SSJ337 cDNA labeled with DIG as a probe, indicated that a single band of 6.3 kb in the wild-type AX2 shifted to 7.7 kb in transformants. In addition, a single band of 3.1 kb in the wild-type AX2 shifted to 4.5 kb in transformants (
To analyze the function of the SSJ337 cDNA clone, SSJ337KO cells were allowed to develop on agar plates, and their morphology was observed. Until the formation of a tight aggregation during development, there were no differences between wild-type AX2 and SSJ337KO cells (Figures 3(a) and (b)). After the post-aggregation stage, the development of SSJ337KO cells became much slower than that of wild-type AX2 cells. Migrating slugs of wild-type AX2 cells appeared at 16 h and mature fruiting bodies formed at 24 h (Figures 3(c) and (e)), while SSJ337KO cells reached the finger stage at 16 h and formed aberrant and tiny fruiting bodies at 36 h after starvation began (Figures 3(d) and (f)).
Transcript levels of the SSJ337 cDNA clone during development were investigated by Northern blotting analysis. Signals were detected in cells starved for 0 to 24 h (
played an important role in the morphogenesis of late development.
Partial DNA nucleotide sequences of the SSJ337 cDNA clone have been determined by the Dictyostelium cDNA project in Japan. A homology search was conducted to identify the protein encoded by the SSJ337 cDNA clone. The nucleotide sequences of the SSJ337 cDNA clone completely corresponded to those of the protein phosphatase 4 catalytic subunit (PP4C, accession no. AF161253). The phosphorylation of serine/threonine residues on proteins by protein kinases is responsible for the communication of many intracellular signals [
One of authors (M. Y.) was a member of the “Dictyostelium cDNA project team in Japan”. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, No. 18580341, and by “Academic Frontier” Project for Private Universities: matching fund subsidy from Ministry of Education, Culture, Sports, Science and Technology.