The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.When Dictyostelium discoideum amoebae are deprived of nutrients, they initiate a developmental program that results in the formation of a multicellular organism composed of differentiated cell types. The aggregation process that takes place early in development is driven by chemotaxis toward cyclic AMP (cAMP) (6), and one of the earliest events in development is the elaboration of the biochemical apparatus required for chemotaxis toward cAMP (for reviews see references 7, 18, and 23). Soon after starvation, the cells synthesize an adenylate cyclase, a cell surface cAMP receptor, an extracellular phosphodiesterase, and a specific phosphodiesterase inhibitor. These proteins, among others, function coordinately to allow the cells to aggregate by chemotaxis. Cells secrete pulses of cAMP every 5 to 6 min, and neighboring cells respond by moving toward elevated concentrations of cAMP and by emitting cAMP to create a relay. The binding of cAMP to cell surface receptors activates second messenger cascades involving G proteins (27) that lead to a variety of cellular events (for reviews, see references 13 and 20), including the chemotactic response and the regulation of genes specific for development. The cAMP receptor is down regulated and phosphorylated upon constant stimulation by cAMP (25,53), and this results in the adaptation of cellular responses (52). The cyclic nucleotide phosphodiesterase (phosphodiesterase) acts outside the cell to reduce cAMP levels, limiting saturation and down regulation of the receptor. The phosphodiesterase exists in membrane-bound and free extracellular forms (30, 47). Its activity is regulated at the gene level (16) and is controlled at the protein level by a specific inhibitor (15). A m...

We have isolated and characterized a Dictyostrfium discoideum gene (PYRI-3) encoding a multifunctional protein that carries the three first enzymatic activities of the de novo pyrimidine biosynthetic pathway. The PYRI-3 gene is adjacent to another gene of the pyrimidine biosynthetic pathway ( P Y R 4 ) ; the two genes are separated by a 1.5-kb non-coding sequence and transcribed divergently. The PYRI-3 gene is transcribed to form a 7.5-kb polyadenylated mRNA. As with the other genes of the pyrimidine biosynthetic pathway, the PYRI-3 mRNA level is high during growth and decreases sharply during development. We have determined the nucleotide sequence of 63% of the coding region of the PYRI-3 gene. We have identified the activities of the protein encoded by the D . discoideum PYRI-3 gene by comparison of amino acid sequences with the products of genes of known function. The P Y R l -3 genc contains four distinct regions that probably correspond to four domains in thc protein. From the NH2 extremity to the COOH extremity, these domains are: glutamine amidotransferase, carbamoylphosphate synthetase, dihydroorotase and aspartate transcarbamylase. This organization is identical to the one found in the rudimentary gene of Drosophila. The evolutionary implications of this finding are discussed.

The development and cellular differentiation of Dictyostelium discoideum are disrupted in transformants secreting high levels of the cyclic nucleotide phosphodiesterase.

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We have performed a differential screen of a Xenopus egg cDNA library and selected two clones (Cli and C12) corresponding to mRNA which are specifically adenylated and recruited into polysomes after fertilization. Sequence analysis of Cll reveals that the corresponding protein is 67.5% identical (83% similar)

The genes coding for the cyclic nucleotide phosphodiesterase (PD) and the PD inhibitory glycoprotein (PDI) have been cloned and characterized. The PDI gene was isolated as a 1.6 kb genomic fragment, which included the coding sequence containing two small introns and 510 nucleotides of non-translated 5' sequence. From the deduced amino acid sequence we predict a protein with a molecular weight (MW) of 26,000 that, in agreement with previous data, contains 15% cysteine residues. Genomic Southern blot analysis indicates that only one gene encodes the inhibitor. Northern blot analysis shows a single transcript of 0.95 kb. The PDI gene is expressed early in development with little transcript remaining following aggregation. The appearance of PDI mRNA is prevented by the presence of cAMP, but when cAMP is removed the transcript appears within 30 minutes. When cAMP is applied to cells expressing PDI the transcript disappears with a half-life of less than 30 minutes. The PD gene of D. discoideum is transcribed into three mRNAs: a 1.9 kb mRNA specific for growth, a 2.4 kb mRNA specific for aggregation, and a 2.2 kb mRNA specific for late development. The 2.2 kb mRNA is also specific for prestalk cells, and is induced by differentiation-inducing factor. All three mRNAs contain the same coding sequence, and differ only in their 5' non-coding sequences. Each mRNA is transcribed from a different promoter, and by using the chloramphenicol acyltransferase gene as a reporter, we have shown that each promoter displays the same regulation as its cognate mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.