Developmental signal transduction pathways uncovered by genetic suppressors - PubMed (original) (raw)

Comparative Study

Developmental signal transduction pathways uncovered by genetic suppressors

G Shaulsky et al. Proc Natl Acad Sci U S A. 1996.

Abstract

We have found conditions for saturation mutagenesis by restriction enzyme mediated integration that result in plasmid tagging of disrupted genes. Using this method we selected for mutations in genes that act at checkpoints downstream of the intercellular signalling system that controls encapsulation in Dictyostelium discoideum. One of these genes, mkcA, is a member of the mitogen-activating protein kinase cascade family while the other, regA is a novel bipartite gene homologous to response regulators in one part and to cyclic nucleotide phosphodiesterases in the other part. Disruption of either of these genes results in partial suppression of the block to spore formation resulting from the loss of the prestalk genes, tagB and tagC. The products of the tag genes have conserved domains of serine protease attached to ATP-driven transporters, suggesting that they process and export peptide signals. Together, these genes outline an intercellular communication system that coordinates organismal shape with cellular differentiation during development.

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Figures

Figure 1

Figure 1

Sequence analysis of mkcA and_regA_. (A) Sequenced regions of_mkcA_ and regA genomic DNA are shown. Coding regions are indicated in boxes, and V shapes indicate introns. Plasmid (4.5 kb, not to scale) insertion sites (IS) in the respective mutant strains are indicated as triangles. Solid box in_mkcA_ is the kinase homology domain. Thatched boxes in_regA_ encode the response regulator homology domain, and checked boxes encode the cyclic nucleotide phosphodiesterase homology domain. Gray boxes under the genes indicate cDNA probes. (B) Sequence similarity between the putative protein kinase domain of_mkcA_ and those of the mitogen-activating protein (MAP) kinase cascade genes PAK65 (Swiss-Prot Protein Sequence Data Bank no. P35465P35465) and STE20 (GenBank accession no. L04655L04655). (C) Sequence similarity between the putative response regulator domain of regA and those of the bacterial response regulators cheY [National Center for Biotechnology Information (NCBI) no. 145525], ntrC (Swiss-Prot Protein Sequence Data Bank no. P10576P10576), and_pleD_ (NCBI no. 1119215). (D) Sequence similarity between the putative cyclic nucleotide phosphodiesterase domain of regA and those of a rat phosphodiesterase (r.PDE; NCBI no. 436012) and a bovine calmodulin stimulated phosphodiesterase (b.PDE; NCBI no. 533781). Conserved regions are underlined, and amino acid numbers are indicated on the left.

Figure 2

Figure 2

Expression of cell type-specific genes in_mkcA_ null or regA null strains. (A) RNA from wild-type cells and from mutants was prepared at 0, 12, and 24 h of development as indicated. Northern blots were hybridized with cotB, a prespore-specific gene probe and with ecmA and ecmB, two prestalk-specific gene probes. (B) Wild-type or mutant cells were developed to early culmination and subjected to in situ RNA hybridization with riboprobes for ecmA,ecmB, and cotB as indicated. Arrow shows the lower cup. (Bar = 0.2 mm.)

Figure 2

Figure 2

Expression of cell type-specific genes in_mkcA_ null or regA null strains. (A) RNA from wild-type cells and from mutants was prepared at 0, 12, and 24 h of development as indicated. Northern blots were hybridized with cotB, a prespore-specific gene probe and with ecmA and ecmB, two prestalk-specific gene probes. (B) Wild-type or mutant cells were developed to early culmination and subjected to in situ RNA hybridization with riboprobes for ecmA,ecmB, and cotB as indicated. Arrow shows the lower cup. (Bar = 0.2 mm.)

Figure 3

Figure 3

Regulation and cell type specificity of_mkcA_ and regA expression. (A) RNA was prepared from developing wild-type cells at 4-h intervals and analyzed by Northern blot analysis using cDNA riboprobes for_mkcA_ or regA (Fig. 1) as indicated. Time (h) is indicated above the lanes; size (kb) is indicated between blots. (B) Wild-type cells were developed to the finger stage or to early culmination and subjected to in situ RNA hybridization with cDNA riboprobes for mkcA or_regA_ as indicated. (Bar = 0.2 mm.)

Figure 3

Figure 3

Regulation and cell type specificity of_mkcA_ and regA expression. (A) RNA was prepared from developing wild-type cells at 4-h intervals and analyzed by Northern blot analysis using cDNA riboprobes for_mkcA_ or regA (Fig. 1) as indicated. Time (h) is indicated above the lanes; size (kb) is indicated between blots. (B) Wild-type cells were developed to the finger stage or to early culmination and subjected to in situ RNA hybridization with cDNA riboprobes for mkcA or_regA_ as indicated. (Bar = 0.2 mm.)

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