G alpha 3 regulates the cAMP signaling system in Dictyostelium.

The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein alpha-subunit G alpha 3 (g alpha 3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of G alpha 3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the g alpha 3- mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the g alpha 3- cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in g alpha 3- cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in g alpha 3- cells starved in the presence of exogenous cAMP pulses. These results suggest that G alpha 3 is essential in early development because it controls the expression of components of the transmembrane signaling system.

Regulation of Dictyostelium early development genes in signal transduction mutants.

The secreted cyclic nucleotide phosphodiesterase (PDE) and its glycoprotein inhibitor (PDI) are among the first genes expressed when Dictyostelium amoebae begin their development. We used a series of mutants with defects in signal transduction to ask whether cAMP receptors 1 and 3, G alpha2, G beta, adenylyl cyclase (ACA), or the protein kinase A catalytic subunit (PKAcat) are required for the initial appearance or later regulation of the PDE and the PDI transcripts. The PDE gene produces a 1.9-kb transcript during vegetative growth and then a 2.4-kb transcript during the early hours of development. Regulation of the 2.4-kb transcript in CAR1, G alpha2, G beta, and ACA mutants is similar to that of isogenic parental strains, although its level is reduced in strains that carry the CAR1 mutation. CAR1/CAR3 double mutants also produce less PDE transcript, but the PDE gene remains inducible by cAMP. The PKAcat mutant produces the 2.4-kb PDE transcript, but in this mutant the vegetative transcript is retained in development. CAR1 and CAR3 are not required for transcription of the PDI gene, but deleting CAR1 leads to overproduction of the PDI transcript. Induction or repression of the PDI gene does not require G alpha2, G beta, or ACA. PKAcat is required for synthesis of the PDI transcript. The results suggest a two-stage regulation of these early genes through a G alpha2/G beta-independent mechanism and an absolute dependence of PDI on the PKAcat.

The phosphodiesterase secreted by prestalk cells is necessary for Dictyostelium morphogenesis.

Dictyostelium discoideum secretes a cyclic nucleotide phosphodiesterase to control cAMP levels during development. Three promoters control expression of the gene--one during vegetative growth, one during aggregation, and one which constrains phosphodiesterase synthesis to prestalk cells. In this report we show that the expression of phosphodiesterase (PDE) in prestalk cells is necessary for morphogenesis. A gene that codes for a specific glycoprotein inhibitor of the phosphodiesterase (Kd = 0.1 nM) was fused to the prestalk-specific promoter of the PDE gene. Transformants carrying multiple copies of this construct secreted inhibitor in 100-fold excess after the aggregation process had occurred. The first effect seen was an elongated tip, followed by a block in slug formation and an inability to culminate. Stalk and spores cells are produced but morphogenesis is uncoupled from cellular differentiation. Overproduction of inhibitor during earlier stages delayed aggregation, but did not affect fruiting body formation. A phosphodiesterase mutant was transformed with a plasmid that expresses PDE only during aggregation and not in prestalk cells. The defect in aggregation was rescued, but the defect in later development was not. The combined results indicate that PDE expression in prestalk cells is critical to morphogenesis. To ask whether the inhibitor gene under its normal regulation had a role in aggregation or later morphogenesis, it was destroyed by homologous recombination. The loss of the gene did not prevent development under the conditions used.

Small circular plasmids of the eukaryote Dictyostelium purpureum define two novel plasmid families.

Two novel groups of circular, nuclear plasmids were discovered in the simple eukaryote Dictyostelium purpureum. They define two new Dictyostelium plasmid families each containing three members: Dpp1A, Dpp1B, and Dpp2 in the Dpp1 family, and Dpp3, Dpp4, and Dpp5 in the Dpp3 family. These plasmids are among the smallest known, ranging in size from 1309 bp (Dpp1A and Dpp1B) to 1961 bp (Dpp4). Family members are very similar. The most distantly related members of the Dpp1 family (Dpp1A and Dpp2) are 89% identical, while the most distantly related members of the Dpp3 family (Dpp3 and Dpp4) are 91% identical. No sequence similarity is found between these plasmid families, or to any other known plasmid or chromosomal DNA sequence. A 72-bp inverted repeat present in one copy in Dpp1A and Dpp1B is tandemly repeated in Dpp2. The Dpp3 family contains a region of 102-160 nucleotides rich in short, overlapping direct sequence repeats. This region is present once in Dpp3 and Dpp5 and is tandemly repeated in Dpp4. The repeat structures in the Dpp1 and Dpp3 families are relatively rich in GC base pairs (29-41%) in comparison to the unique sequence regions of the plasmids (16-22%). The longest open reading frame (ORF) beginning with an AUG codon in these plasmids is 168 bp in the Dpp3 family, although longer ORFs without AUG start codons (up to 201 bp) also exist. Northern blot analysis did not detect any plasmid-specific transcripts in total RNA prepared from vegetative cells carrying Dpp1A or Dpp3.

Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418.

A protocol that allows the rapid isolation and growth of large numbers of independent G418-resistant Dictyostelium discoideum transformant colonies on the surface of agar media with live bacteria was developed. Transformants grown under these conditions form normal fruiting bodies. Discovery that aggregation of nontransformants was inhibited at a nonselective level of G418 (25 to 35 micrograms/ml) led to the development of a vector maintenance assay. Using this assay we examined the stability of recombinant plasmids derived from the D. discoideum native plasmids Ddp1 and Ddp2. We conclude that the origin of replication of plasmid Ddp1 does not alone confer stable maintenance and thus, Ddp1 must bear additional sequences required for its own maintenance. Analysis of the maintenance of vectors derived from Ddp2 showed that autonomously replicating shuttle vectors that contained bacterial plasmid DNA and from which one element of the Ddp2 inverted repeat was removed were much less stable than vectors that contained a complete inverted repeat or that did not carry a bacterial plasmid. Sequences between the 3' end of the rep gene and the inverted repeat appear to play a role in plasmid maintenance. An intact rep gene and one copy of the inverted repeat element were required for extrachromosomal replication. Maintenance of extrachromosomal vectors was found to be strain dependent. Four traits distinguishing integrating vectors from those capable of autonomous replication were identified.

Cyclic nucleotide phosphodiesterase of Dictyostelium discoideum and its glycoprotein inhibitor: structure and expression of their genes.

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)

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development.

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.

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum utilizes alternate promoters and splicing for the synthesis of multiple mRNAs.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) gene plays essential roles in the development of Dictyostelium discoideum during cellular aggregation and postaggregation morphogenesis. Genomic clones spanning the gene were isolated and used to determine the sequence and structure of the phosphodiesterase gene. We found an unusually complex organization for a gene of D. discoideum. Two transcripts of 2.4 and 1.9 kilobases (kb) were synthesized from start sites separated by 1.1 kb. A developmentally regulated promoter was utilized for the 2.4-kb mRNA, and a constitutive promoter regulated synthesis of the 1.9-kb transcript. The gene was found to be divided into four exons that are alternately spliced to give rise to the two mRNAs. The precursor of the 2.4-kb mRNA contained a 2.3-kb intron, whereas the precursor of the constitutive transcript was synthesized with a 1.7-kb intron. The two transcripts contained identical protein-coding regions and 400-nucleotide 3' untranslated sequences. The 2.4-kb developmentally regulated mRNA was distinguished by a long 5' untranslated leader of 666 nucleotides. The complex structure of the gene may allow multiple levels of control of the expression of the phosphodiesterase during development.

Rescue of a Dictyostelium discoideum mutant defective in cyclic nucleotide phosphodiesterase.

One of the developmentally induced gene products that is essential for chemotaxis of Dictyostelium amoebae is a cyclic nucleotide phosphodiesterase. The enzyme can be secreted or exist in a membrane bound form. This enzyme is missing in the mutant HPX235 which, as a consequence, does not aggregate unless exogenous cAMP phosphodiesterase is supplied. We have introduced multiple copies of the cloned phosphodiesterase gene into mutant amoebae and restored aggregation. The formation of anatomically correct fruiting bodies, which does not occur when exogenous enzyme is added, is also restored by transformation with the gene. The construct that we have used gives rise only to secreted phosphodiesterase and therefore the membrane bound form of the enzyme is not absolutely required for normal aggregation and morphogenesis.

Disruption of Dictyostelium discoideum morphogenesis by overproduction of cAMP phosphodiesterase.

The development and cellular differentiation of Dictyostelium discoideum are disrupted in transformants secreting high levels of the cyclic nucleotide phosphodiesterase. The aggregation of these cells in the early stage of development proceeds rapidly and without the formation of organized streams. The later stages of development, in which differentiation into stalk and spore cells normally takes place, are completely blocked so that the transformants remain in spherical clusters of undifferentiated cells that do not elaborate the tip structure that regulates morphogenesis. These effects are due to overproduction of extracellular phosphodiesterase and demonstrate the role of cAMP during the aggregation phase of development as well as in the control of differentiation and pattern formation.

The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum: the structure of the gene and its regulation and role in development.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum plays an essential role in development by hydrolyzing the cAMP used as a chemoattractant by aggregating cells. We have studied the biochemistry of the phosphodiesterase and a functionally related protein, the phosphodiesterase inhibitor protein, and have cloned the cognate genes. A 1.8-kb and a 2.2-kb mRNA are transcribed from the single-phosphodiesterase gene. The 2.2-kb mRNA comprises the majority of the phosphodiesterase mRNA found in differentiating cells and is transcribed only during development from a promoter at least 2.5 kb upstream of the translational start site. The 1.8-kb phosphodiesterase mRNA is detected at all stages of growth and development, is present at lower levels than the developmentally induced mRNA, and is transcribed from a site proximal to the protein-coding region. The phosphodiesterase gene contains a minimum of three exons, and a 2.3-kb intron, the longest yet reported for this organism. We have shown that the pdsA gene and four fgd genes affect the accumulation of the phosphodiesterase mRNAs, and we believe that these loci represent a significant portion of the genes regulating expression of the phosphodiesterase. The phosphodiesterase gene was introduced into cells by transformation and used as a tool to explore the effects of cAMP on the terminal stages of development. In cells expressing high levels of phosphodiesterase activity, final morphogenesis cannot be completed, and differentiated spore and stalk cells do not form. We interpret these results to support the hypothesis that cAMP plays an essential role in organizing cell movements in late development as well as in controlling the aggregation of cells in the initial phase of the developmental program.

The expression of two transcripts of the phosphodiesterase gene during the development of Dictyostelium discoideum.

One of the earliest events in the development of Dictyostelium discoideum is the induction of the cyclic nucleotide phosphodiesterase gene. During vegetative growth a small amount of secreted phosphodiesterase is synthesized. The phosphodiesterase transcript which is responsible for the vegetative enzyme has a size of 1800 nucleotides. Soon after starvation begins a more abundant mRNA with a size of 2200 nucleotides is synthesized by the developing cells. The induction of the 2200-nucleotide mRNA is dependent on protein synthesis and takes place under all regimens of growth and starvation. When growth is in axenic medium and development is in phosphate buffer, the appearance of the larger transcript is very rapid, occurring within 30 min after the onset of starvation. The initial burst of phosphodiesterase mRNA synthesis is followed by a decline in mRNA abundance unless the cells are stimulated by cAMP. When cells are grown on bacteria and development takes place on filter paper, the larger transcript appears after 4 hr, reaches a peak at 10-12 hr of development, and then slowly disappears. When prestalk and prespore cells from migrating slugs are separated, a small amount of transcript can be found only in the prestalk cells. A series of mutants blocked early in development make very little phosphodiesterase transcript or are otherwise abnormal in expression of the phosphodiesterase mRNA. Together these mutants define five independent genetic loci which affect the accumulation of the phosphodiesterase mRNA. These are the pdsA, fgdA, fgdC, fgdD, and fgdE genes.

Molecular cloning and developmental expression of the cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum.

The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum functions to maintain the responsiveness of cells to the chemoattractant cAMP during the aggregation phase of development. We have prepared a cDNA library and have isolated clones which contain a portion of the 5' untranslated region and the entire coding and 3' untranslated portions of the cyclic nucleotide phosphodiesterase gene. The primary structure of the extracellular cyclic nucleotide phosphodiesterase precursor has been deduced from the nucleotide sequence. The molecule is composed of 452 amino acids and was calculated to have a molecular mass of 51,078 daltons. Forty-nine amino-terminal residues which contain a hydrophobic leader sequence are not present in the mature extracellular enzyme. Four potential asparagine-linked glycosylation sites were found within the phosphodiesterase. An amino acid sequence homology search revealed no closely related proteins. Phosphodiesterase mRNA levels are low in growing cells and first increase soon after the onset of development. The amount of transcript then decreases before rising in abundance to maximum levels during the terminal stages of cell aggregation and apical tip formation. During formation of the fruiting body, levels of phosphodiesterase mRNA decrease. Exposure of cells to cAMP increases the amount of phosphodiesterase mRNA. Increases of mRNA abundance are correlated with increases in enzyme activity, suggesting regulation at the level of transcription.

Isolation of a cDNA encoding a portion of the cyclic nucleotide phosphodiesterase of Dictyostelium discoideum.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum is one of a group of developmentally regulated proteins which enable cells to aggregate by chemotaxis during the early stages of development. We report the identification and DNA sequence of a cDNA clone encoding the amino-terminal region of the phosphodiesterase. The clone, pPD-3, was selected from a cDNA library created by priming first strand synthesis using a set of oligonucleotides with sequences predicted from the amino-terminal amino acid sequence of purified phosphodiesterase. The DNA sequence of pPD-3 encodes perfectly the available phosphodiesterase amino acid sequence, and pPD-3 selects an mRNA which can be translated into material recognized by phosphodiesterase antisera. The nucleotide sequence of pPD-3 indicates there are 49 amino acids, which contain a segment possessing the characteristics of a signal peptide, that separate the amino-terminal residue identified in the purified protein from the methionine codon at which translation originates. DNA blot analysis demonstrates that the phosphodiesterase gene exists as a single copy in the nuclear genome. Analysis of RNA indicates that the phosphodiesterase transcript is 2.1 kb long, which is approximately 0.8 kb more than the minimum required to encode this protein.