Isolation and analysis of a developmentally regulated gene of dictyostelium discoideum

Dictyostelium discoideum, a cellular slime mold, is a free-living amoeba that lives in the surface layer of soil and forest litter. The cells feed on a variety of bacteria including Escherichia coli and Aerobacter aerogenes. In the vegetative phase of its life cycle, D. discoideum lives as solitary free-living amoeba. Upon starvation, growth is terminated and development is initiated resulting in the expression of many new genes (reviewed in Kessin, 1988). This eukaryotic microorganism goes through distinct phases of growth, starvation, aggregation, differentiation, and finally the germination of spores to restart the whole cycle over again (Blumberg, 1991 ). In order to study developmentally regulated genes, a polyclonal antiserum was raised against differentially expressed antigens in developing fruiting bodies of D. discoideum using an antiidiotypic inoculation method (Barclay and Smith, 1986). A 500 bp eDNA, Dicty6, was isolated. This eDNA fragment was used as a probe for D. discoideum RNA and genomic DNA blot hybridizations. This probe recognizes a single, developmentally regulated mRNA species about 1.2 kb in length. Total cellular RNA was extracted from NC-4 cells during development from 0-24 hours and used in RNA blots. Dicty6 mRNA accumulates at about 8 hours, gradually increases, and peaks at 18-20 hours. The expression of this mRNA is influenced by cAMP and DIF (differentiation inducing factor)-a morphogen that induces stalk cell development. DIF induction experiments revealed that Dicty6 is repressed by DIF since Dicty6 mRNA accumulates to higher levels in DIFcultures than DIF+ cultures. Since sequencing revealed that the isolated cDNAs are not full length, a bacterial D. discoideum eDNA library was screened. A 1 kb eDNA was isolated. Comparison of nucleotide sequence and derived amino acid sequence using GenBank EMBL databases revealed that this gene is unique. Genomic DNA hybridizations indicate that this mANA is the product of a single gene in the D. discoideum genome. A 4.5 kb genomic clone was isolated. Sequencing data revealed that the genomic clone contains the whole coding sequence with no introns. It also contains another 3.5 kb at the 5• untranslated region.