ABSTRACT
We isolated and characterized a gene from Dictyostelium discoideum, which encodes a protein of 279 amino acids (30.6kDa) containing six transmembrane domains with two highly conserved motifs of asparagine-proline-alanine (NPA) found in the aquaporin family of water-channel proteins, although the second motif of the protein has been modified into NPV (asparagine-proline-valine). The deduced amino acid sequence of the gene, which we have named aqpA, is 39% identical to D. discoideum WacA, 26% identical to human Aqp5, 26% identical to Oryza sativa PIP2a, 25% identical to yeast Aqy1 and 24% identical to E.coli AqpZ. Southern analyses indicated that aqpA is present as a single copy in the genome. Northern blot analysis showed that the developmentally regulated 1kb mRNA transcript first appears at the tight mound stage (12h), and is abundant in fingers (16h) and late culminants (20h). In-situ hybridization of slugs revealed that aqpA mRNA accumulated in cells of the prespore region but not in those of the prestalk region. Disruption of aqpA by homologous recombination did not significantly affect growth or developmental morphogenesis. Although mutant spores were viable, when assayed soon after encapsulation, they became permeable to propidium iodide and lost viability after a week on the top of a fruiting body. Thus, AqpA is essential to maintain spore dormancy perhaps through the regulation of water flow.
Subject(s)
Aquaporins/genetics , Dictyostelium/genetics , Protozoan Proteins , Amino Acid Sequence , Animals , Base Sequence , Cell Division/genetics , DNA/chemistry , DNA/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Dictyostelium/cytology , Dictyostelium/physiology , Gene Expression Regulation, Developmental , Microscopy, Fluorescence , Molecular Sequence Data , Mutagenesis , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Spores/physiologyABSTRACT
In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.
