織井 秀文

In fresh water planarian Dugesia japonica, two genes encoding POU-domain proteins, DjPOU1 and DjPOU2, have been identified by polymerase chain reaction (PCR). Their POU-domains are strikingly similar to class III POU-domains in mammals, frog, insects and nematode. The full-length cDNA of DjPOU1 has been cloned and sequenced, which revealed that it codes for a protein of 559 amino acids. The accumulation of DjPOU1 mRNA is two or three times more in anterior region of worm containing a brain, than the other regions, and decreases during regeneration after amputation.

The Dp87 is a novel prespore specific gene of Dictyostelium discoideum which has a long open reading frame of 555 amino acids. The entire amino acid sequence had low but significant homology to the spore coat proteins, SP96 and SP70, of this organism. When a chimeric gene, containing a 1380 bp of the 5' upstream region of this gene fused with CAT gene, as reporter, was introduced into cells of this organism, it was expressed only in prespore cells of the slug. Transformation experiments, using chimeric genes, containing a series of 5' deletions of the upstream region, showed that -447 bp to -357 bp is an important cis-acting regulatory region for transcription. A nuclear factor(s) that specifically bind to this cis-acting region were detected from slug cell nuclei. Transformation experiments using a chimeric gene consisting of the 5' region between -666 bp and +149 bp of this gene, a beta-galactosidase reporter and an actin 8 terminator, showed that the reporter gene was expressed as early as in aggregation streams, indicating that Dp87 become transcribed a few hours earlier than the other prespore-specific genes so far reported. This was confirmed by northern hybridization detected using an image plate analyzer. The fact that cells expressing Dp87 appeared at random in aggregation streams gives solid support to the idea that position-independent differentiation of prespore and prestalk cells, followed by their sorting, brings about pattern formation in this organism.

The spore-coat protein gene (SP96) of Dictyostelium discoideum is transcribed only in prespore cells. To identify the cis-acting region of this gene, mutant mini-genes which contained different lengths of 5' upstream region, the partially deleted SP96 coding region and ca. 600 bp of 3' flanking sequence were transformed into D. discoideum cells. Expression of the mini-genes was analysed by Northern hybridization. Our results indicate that the 5' upstream region from -686 to -494 contains an important cis-acting element for the temporal and cell type-specific transcription. A nuclear factor which specifically bound the cis-acting region was identified by gel retardation assay. DNase-I-hypersensitivity of the 5' upstream region was examined and it was shown that the appearance of two new hypersensitive sites correlates with transcriptional activation of the gene. One of the two sites maps to the TATA region and the other was located in the cis-acting region identified by deletion analysis. Our results suggest that gene activation occurs by conformational changes in the chromatin structure of the cis-acting region followed by subsequent binding of regulatory factors and the TATA-binding protein.

We have determined the complete nucleotide sequence of pDG1, a plasmid found in a wild isolate of Dictyostelium. The 4439-bp long pDG1 contains only one, 2718-bases-long, open reading frame (ORF) and nearly perfect inverted repeats of 551 bp and 552 bp. Northern-blot analysis showed that only one 2.7-kb poly (A)+ RNA transcript was expressed at a maximum level, 2 h (early aggregation stage) after the onset of development. The expression of this transcript was suppressed by the addition of cAMP. In the upstream region of the ORF, there are several putative consensus sequences, e.g. (1) TGACTTAGAA-AAATT which is a putative site for cleavage by topoisomerase I, and (2) TGACGACA which may be a cAMP-responsive element, found in several genes that are regulated by cAMP at the level of transcription. A possible mechanism of the partitioning of pDG1 into daughter cells is discussed.

We constructed a recombinant plasmid containing the 2.1 kb HindIII fragment of plasmid pDG1, isolated from the cellular slime mold (Dictyostelium sp. strain GA11), and using pAG60 as cloning vector. We found that deletions of the recombinant plasmid took place frequently in Escherichia coli wild-type cells. However, the deletion was not observed when the plasmid was introduced into a strain that was an isogenic temperature-sensitive mutant of the gyrA gene. These results suggest that E. coli DNA gyrase is involved in the mechanisms of the deletion formation. It was shown that the 1.0 kb deletant derived from the 2.1 kb HindIII insert was produced by elimination of a 1.1 kb region. Sequence analysis of the deletants showed that cutting and rejoining took place between two out of the six nearly perfect direct repeats [21 bp palindromic sequences; AAAAAA(T/C)GGC(G/C)GCC(A/G)TTTTTT], located near the distal ends of the inverted repeats, preserving one copy of the repeats. These sequences consist of local short inverted repeats, where cutting and rejoining occur at one of the two regions.

A circular plasmid having high copy number was found in a wild isolate of Dictyostelium species. Gel electrophoresis, electron microscopy and Southern blot hybridization revealed that the plasmid, named pDG1, is 4.5 Kb (1.5 micron) in size with closely situated long inverted repeats. The plasmid seems to be located in the nuclei. It was not a derivative of ribosomal DNA. The possible correlation of the plasmid with the putative intermediate DNA of retrotransposon DIRS-1 found in Dictyostelium discoideum is discussed.

In Dictyostelium discoideum cyclic AMP (cAMP) metabolism during macrocyst development, i.e., the sexual cycle of this organism, and in giant cells, i.e., fusion products from opposite mating-type cells, was investigated. The pattern of change in cAMP levels during macrocyst development differed considerably from that observed during fruiting-body formation, i.e., the asexual cycle. Giant cells produced and excreted considerable amounts of cAMP. Adenylate cyclase activity catalyzing cAMP production in giant cells was comparable to that of unfused cells. However, the activity of membrane-bound phosphodiesterase in giant cells was extremely low, and no extracellular phosphodiesterase was excreted. A phosphodiesterase inhibitory protein was secreted in excess by giant cells.