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. 1998 Jun 1;12(11):1738-48.
doi: 10.1101/gad.12.11.1738.

A novel Myb homolog initiates Dictyostelium development by induction of adenylyl cyclase expression

H Otsuka  1 , P J Van Haastert
Affiliations

A novel Myb homolog initiates Dictyostelium development by induction of adenylyl cyclase expression

H Otsuka et al. Genes Dev. .

Abstract

Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis we have isolated a mutant that does not aggregate upon starvation but is rescued by adding extracellular cAMP. Sequencing of the mutated locus revealed a new gene, DdMYB2, whose product contains three Myb repeats, the DNA-binding motif of Myb-related transcription factors. Ddmyb2-null cells show undetectable levels of ACA transcript and no cAMP production. Ectopic expression of ACA from a constitutive promotor rescues differentiation and morphogenesis of Ddmyb2-null mutants. The results suggest that development in Dictyostelium starts by starvation-mediated DdMyb2 activation, which induces adenylyl cyclase activity producing the differentiation-inducing signal cAMP.

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Figures

Figure 1
Figure 1
The DdMYB2 locus and isolation of a Ddmyb2–null mutant. (A) REMI and gene targeting at DdMYB2 locus. W67 mutant was made by REMI of the BamH1-linearized plasmid pJB1 into the genome of uracil–auxothroph wild-type DH1 with the restriction enzyme DpnII. The DdMYB2 locus was isolated from genomic DNA using the restriction enzyme BglII. The Ddmyb2-null mutant was made by homologous recombination in wild-type AX3 genome with pMKO1, which contains a BSR, and pBluescript at the SacI site of the NheI–BglII genomic DNA fragment. The expected sizes of BglII fragments from wild-type cells and Ddmyb2 disruptant are indicated. (B) Southern analysis of the transformants with pMKO1. DNA was isolated from randomly picked transformants, digested with BglII, and probed with the SacI–BglII genomic fragment containing 3′ portion of DdMYB2 gene. MD255 was used as Ddmyb2 disruptant for further analysis. (C) Expression of DdMYB2 in wild-type AX3 and in Ddmyb2–null mutant MD255. RNA was prepared from either growing cells or cells that had been starved in suspension for the time indicated. The blot was probed with partial cDNA for DdMYB2 RNA (nucleotides 1675–1974 in Fig. 2B).
Figure 2
Figure 2
A novel myb-related gene DdMYB2. (A) Nucleotide and deduced amino acid sequence of the DdMYB2 gene. Putative TATA-oligo(dT) and polyadenylation signals are underlined. (arrow) The putative start site of transcription, (*) the end of a cDNA isolate. The deduced amino acids sequence from 425 to 592 (boxed) consists of three Myb repeats. (B) Alignment of Myb domains from various Myb-related proteins. (D.d.) Dictyostelium discoideum Myb2 (this study), Myb1 (or MybH; Stober-Gräser et al. 1992); (H.s.) Homo sapiens c-Myb (Majello et al. 1986); (S.p.) Shizosacharomyces pombe Cdc5 (Ohi et al. 1994), (D.m.) Drosophila melanoguster c-Myb (Katzen et al. 1985); (Z.m.) Zea mays P (Grotewold et al. 1991). Boxes indicate amino acid identity between DdMyb2 and other proteins. Secondary structure prediction using PHD is shown for DdMyb2: (−) loop, (□) helix, (.) no prediction. The c-Myb helices determined by nuclear magnetic resonance (NMR) are indicated with shaded boxes (Ogata et al. 1994).
Figure 2
Figure 2
A novel myb-related gene DdMYB2. (A) Nucleotide and deduced amino acid sequence of the DdMYB2 gene. Putative TATA-oligo(dT) and polyadenylation signals are underlined. (arrow) The putative start site of transcription, (*) the end of a cDNA isolate. The deduced amino acids sequence from 425 to 592 (boxed) consists of three Myb repeats. (B) Alignment of Myb domains from various Myb-related proteins. (D.d.) Dictyostelium discoideum Myb2 (this study), Myb1 (or MybH; Stober-Gräser et al. 1992); (H.s.) Homo sapiens c-Myb (Majello et al. 1986); (S.p.) Shizosacharomyces pombe Cdc5 (Ohi et al. 1994), (D.m.) Drosophila melanoguster c-Myb (Katzen et al. 1985); (Z.m.) Zea mays P (Grotewold et al. 1991). Boxes indicate amino acid identity between DdMyb2 and other proteins. Secondary structure prediction using PHD is shown for DdMyb2: (−) loop, (□) helix, (.) no prediction. The c-Myb helices determined by nuclear magnetic resonance (NMR) are indicated with shaded boxes (Ogata et al. 1994).
Figure 3
Figure 3
Phenotype of the Ddmyb2-null mutant and rescue by exogenous cAMP pulses or by ectopic expression of ACA. Cells starved in phosphate buffer for 5 hr were plated on nonnutrient agar at a density of 2 × 106/cm2. The photographs were taken after 30 hr; overhead views at left; side views at right. (A) No aggregation of Ddmyb2-null cells; (B) Wild-type fruiting bodies; development and fruiting body formation are restored by addition of exogenous cAMP pulses to Ddmyb2-null cells during 5 hr of starvation (C) or by constitutive expression of ACA (D). Scale bar is 1 mm.
Figure 4
Figure 4
Defective signal transduction in Ddmyb2-null cells and its rescue by cAMP pulses. Cells were starved in suspension and pulsed with or without 100 nm cAMP added every 5 min. Wild-type Ax3 without pulses (□); Ddmyb2–null cells without (○) or with pulses (•). (A) Cells were starved with or without cAMP pulses for the indicated time and assayed for cAMP-binding activity. (B) cAMP-induced cGMP response, (C) cAMP relay response, and (D) adenylyl cyclase activity in wild-type AX3 cells starved for 5 hr or Ddmyb2–null cells starved for 7 hr. Adenylyl cyclase activity was measured in cell lysates with Mn2+–ATP as substrate.
Figure 4
Figure 4
Defective signal transduction in Ddmyb2-null cells and its rescue by cAMP pulses. Cells were starved in suspension and pulsed with or without 100 nm cAMP added every 5 min. Wild-type Ax3 without pulses (□); Ddmyb2–null cells without (○) or with pulses (•). (A) Cells were starved with or without cAMP pulses for the indicated time and assayed for cAMP-binding activity. (B) cAMP-induced cGMP response, (C) cAMP relay response, and (D) adenylyl cyclase activity in wild-type AX3 cells starved for 5 hr or Ddmyb2–null cells starved for 7 hr. Adenylyl cyclase activity was measured in cell lysates with Mn2+–ATP as substrate.
Figure 5
Figure 5
Gene expression of cAMP-signaling components in Ddmyb2-null cells. Wild-type AX3 and Ddmyb2–null cells were starved in suspension with or without cAMP pulses for the indicated time. Total mRNA was isolated and analyzed. The Northern blot shows the expression of CAR1, ACA, and Gα2.
Figure 6
Figure 6
Restoration of cAMP oscillations in Ddmyb2-null cells by ectopic expression of ACA. Cells were starved for 5 hr, washed, and resuspended to a density of 108 cell/ml. Samples of the cell suspension were taken at 1-min intervals and used to determine the cAMP concentration. (A) Wild-type Ax3; (B) Ddmyb2–null cells (○), Ddmyb2−/ACA cells (•). The results shown are from a typical experiment reproduced two times.
Figure 7
Figure 7
Model of the regulation of early development in Dictyostelium by Myb2.
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