Ribosomal protein gene expression is cell type specific during development in Dictyostelium discoideum.

Starvation for amino acids initiates the developmental cycle in the cellular slime mold, Dictyostelium discoideum. Upon starvation one of the earliest developmental events is the selective loss of the ribosomal protein mRNAs from polysomes. This loss depends upon sequences in the 5' non-translated leader of the ribosomal protein (r-protein) mRNAs. Here evidence is presented which indicates that those cells which will become prestalk cells express the ribosomal protein genes during development under starvation conditions. Cells which enter the prespore pathway shut off r-protein synthesis. The promoter and 5' non-translated leader sequences from two ribosomal protein genes, the rp-L11 and the rp-S9 genes, are fused to the Escherichia coli beta-galactosidase reporter gene. While beta-galactosidase enzyme activity is detected in situ in most growing cells, by 15 h of development beta-galactosidase enzyme activity is largely lost from the prespore cells although strong beta-galactosidase enzyme activity is present in the prestalk cells. These observations suggest the possibility that the ribosomal protein mRNAs are excluded from polysomes in a cell-type-specific manner.

Dictyostelium ribosomal protein genes and the elongation factor 1B gene show coordinate developmental regulation which is under post-transcriptional control.

Starvation for amino acids initiates the developmental program in the cellular slime mold, Dictyostelium discoideum [19, 20]. One of the earliest developmental events is the decline in ribosomal protein synthesis [2, 17, 29, 30]. The ribosomal protein mRNAs are excluded from polysomes with 20 min to 1 h following the removal of nutrients, and their mRNA levels decline sharply at about 9 h into the 24-h developmental cycle [28, 31, 35, 36]. It has been generally assumed that the decline in r-protein mRNA levels during late development reflected a decline in the transcription rate [12, 32, 43]. Here we demonstrate that this is not the case. The transcription rates of three ribosomal protein genes, rpL11, rpL23 and rpS9 as well as an elongation factor 1B gene have been determined during growth and development in Dictyostelium. Throughout growth and development the transcription rate of the ribosomal protein genes remains relatively constant at 0.2%-0.5% of the rate of rRNA transcription while the elongation factor 1B gene is transcribed at 0.4%-0.6% of the rRNA rate. This low but constant transcription rate is in contrast to a spore coat protein gene Psp D, which is transcribed at 6% of the rRNA rate in late developing cells. The elongation factor 1B gene appears to be co-regulated with the ribosomal protein genes both in terms of its transcription rate and mRNA accumulation. Dictyostelium has been a popular model for understanding signal transduction and the growth to differentiation transition, thus it is of significance that the regulation of ribosome biosynthesis in Dictyostelium resembles that of higher eukaryotes in being regulated largely at the post-transcriptional level in response to starvation as opposed to yeasts where the regulation is largely transcriptional [27].

Analysis of a novel cyclic Amp inducible prespore gene in Dictyostelium discoideum: evidence for different patterns of cAMP regulation.

The D7 cDNA clone hybridizes to a 2.8 kb mRNA which first appears at the mound stage of development in the cellular slime mold Dictyostelium discoideum. This gene which is cyclic AMP (cAMP) inducible and is expressed specifically in the prespore cells contains an open reading frame interrupted by only one intron. The predicted amino acid sequence indicates a novel prespore protein which differs from all of the previously described prespore proteins in that it contains no internal repeats and does not share any homology with any of the other prespore genes. The amino acid sequence predicts a protein of 850 amino acids with a molecular weight of 95,343 daltons and an isoelectric point of 4.25. The protein is very rich in glutamine (13.8%), asparagine (10.6%) and glutamic acid (10.4%) with one potential glycosylation site and 28 possible sites for phosphorylation. The amino terminus is hydrophobic with characteristics of a signal sequence while the entire carboxyl half of the protein is notable for its hydrophilicity. Comparison of cAMP regulation of the D7 gene with the regulation of two other cAMP regulated prespore genes, the PL3(SP87) gene and the Psa(D19), reveals some striking differences. Disaggregation in the presence of cAMP results in transient degradation of mRNA for all three genes. The transcription rate for the D7 and PsA(D19) genes remains relatively unaffected by disaggregation but there is a rapid although transient decline in the transcription rate of the PL3(SP87) gene. Although the accumulation of all three mRNAs is first detectable at mound stage, transcription of the D7 and PsA(D19) genes is detected earlier in development, at rippling aggregate stage several hours prior to the earliest time when transcription of the PL3(SP87) gene is detected. Analysis of the promoter region of the D7 gene reveals three CA like boxes flanked by direct repeats as well as four G rich regions that may serve as regulatory elements.

The promoter of a gene encoding a novel Dictyostelium spore coat protein.

The cAMP-inducible prespore gene, PL3, encodes a protein which is a novel component of the spore coat. Unlike the well-characterized spore coat proteins (SP60, SP70, and SP96) which are found in the outer layer of the coat, the PL3 gene product is localized to a subregion of the coat beneath the outer proteinaceous layer. Moreover, a substantial portion of the PL3 protein is tightly associated with the spore coat and not released under the conditions that led to the identification of the other coat proteins. The promoter for this novel spore coat gene is described. Unlike the other coat-protein gene promoters, it lacks the extensive CA-type elements. It contains two short CA boxes and five prominent G-rich regions. Sequential deletions from the 5' end of the promoter which remove both CA boxes as well as two of the G-rich regions reduce the level of expression but do not alter the spatial regulation of expression. Despite the sequence differences, the PL3 promoter still confers correct spatial, temporal, and cell type-specific regulation on a reporter gene. Escherichia coli beta-galactosidase enzyme activity expressed under the control of this PL3 promoter first appears in randomly isolated cells at the loose mound stage. Because of the sensitivity of the assay, beta-galactosidase activity is detectable prior to the appearance of the PL3 protein on Western blots and by immunofluorescence. Later the number of cells staining for beta-galactosidase activity and the intensity of staining increases. During tipped mound, slug, and culminant stages, cells expressing beta-galactosidase under the control of the PL3 promoter are localized to prespore regions and are spatially coincident with cells expressing the PL3 protein.

Identification of a new spore coat protein gene in the cellular slime mold Dictyostelium discoideum.

Genomic DNA encoding the prespore cell-specific PL3 cDNA was cloned and sequenced, revealing that the gene consists of three exons separated by short 100-bp introns. The single long open reading frame predicts a primary translation product of 70 kDa after removal of a cleavable signal peptide, two-thirds of which consists of four kinds of amino acid repeat elements, including two found in other spore coat proteins. The 85-kDa PL3 protein synthesized in vivo accumulates specifically in regulated secretory vesicles of prespore cells (prespore vesicles), as determined microscopically using antibody against a PL3 gene fusion product expressed in Escherichia coli. The protein later accumulates extracellularly in the spore coat, which is formed during sporulation, as determined ultrastructurally and by Western blot analysis of SDS-PAGE gels. In addition to its high proportion of repeat elements, the PL3 protein has the following properties which distinguish it from other spore coat proteins: (1) it is located at the outer extent of the middle layer, beneath the outer layer, (2) its dissociation from the coat requires the presence of protein denaturants and reducing agents at elevated temperature, and (3) a large proportion of the protein is not dissociated from the coat even under these conditions, as determined by ultrastructural analysis of the extracted coat. The PL3 protein may contribute to the structure of the coat at the interface between the middle, cellulosic layer and the outer, electron-dense, proteinaceous layer.

Gene expression and chromatin structure in the cellular slime mold, Dictyostelium discoideum.

Micrococcal nuclease digestion of chromatin from growing cells reveals a structural organization which differs for genes transcribed at diverse rates. The late cAMP dependent prespore genes which are not transcribed in growing cells are found in growing cells in a regular nucleosomal repeat with an average spacing of 168 nucleotides. By contrast genes expressed at a low level in growing cells show an irregular pattern of bands with an average distance between bands of 80 nucleotides. The sizes of the bands generated from the transcribed genes are consistent with the concept that transcription results in the loss of the linker region histone H1 with concomitant sliding of nucleosomes to generate close packed ("slipped") di, tri, and tetra nucleosomes lacking the linker region. Further analysis of dinucleosomes released by micrococcal nuclease digestion reveals that transcriptionally active genes are found associated with dinucleosomes species which may be lacking histone H1. The length of DNA protected by these dinucleosomes is heterogeneous, ranging from 250 to 300 nucleotides. Methodology is described which has been adapted to allow two dimensional hybridization mapping of nucleoprotein complexes on single copy Dictyostelium genes.

Ca++ antagonists distinguish different requirements for cAMP-mediated gene expression in the cellular slime mold, Dictyostelium discoideum.

Cyclic AMP is essential for the accumulation of many prespore mRNAs and can advance the time of appearance of mRNAs specifically enriched in prestalk cells. Additionally, when late-developing cells are washed free of cAMP, a number of growth phase mRNAs reaccumulate. This reaccumulation can be suppressed by cAMP. These effects of cAMP are all mediated through the cell surface cAMP receptor and can occur under conditions where the receptor-associated adenylate cyclase is inactive, indicating that the initial intracellular transduction event necessary for expression of these mRNAs does not depend upon cAMP synthesis. The dihydropyridine derivatives, nifedipine and nitrendipine, are highly specific Ca++ channel blockers. They are shown here to prevent the influx of Ca++ from the external medium that occurs in response to cAMP binding to the cell surface receptor during development. These two compounds as well as another Ca++ antagonist, 8-N,N-diethylamino)octyl-3,4,5-trimethoxy-benzoate (TMB-8) and a calmodulin inhibitor, N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide (W7), all specifically decrease cAMP-mediated prespore mRNA accumulation in a dose-dependent manner. They also prevent cAMP from suppressing the expression of the growth phase genes. The growth phase mRNAs reaccumulate in cAMP-treated cells in the presence of increasing concentrations of these drugs. By contrast, cAMP induction of the pre-stalk-enriched mRNA is not as significantly affected by these agents. These results raise the possibility that the cell surface cAMP receptor can couple to different signal transduction systems and thereby induce or suppress the expression of different sets of cAMP-regulated genes during development.

A Ca2+-dependent signal transduction system participates in coupling expression of some cAMP-dependent prespore genes to the cell surface receptor.

Elevated levels of cAMP are essential for the expression of many postaggregation prespore and prestalk mRNA species and for the suppression of some growth phase mRNAs. Here we review evidence that this regulation is mediated by cAMP interacting at the cell surface receptor. These effects of cAMP on gene expression can occur under conditions where the receptor-associated adenylate cyclase is inactivated and in concentrations that are consistent with receptor-binding. A number of differences are noted in the mechanism by which cAMP regulates prespore and prestalk genes. Finally, evidence is reviewed for the role of a Ca2+-dependent signal transduction system in coupling the expression of some of the prespore mRNAs to the cAMP receptor. This signal transduction system does not appear to be involved in the expression of the cAMP-dependent prestalk gene.

Changes during differentiation in requirements for cAMP for expression of cell-type-specific mRNAs in the cellular slime mold, Dictyostelium discoideum.

A number of genes encoding developmentally regulated mRNAs in the cellular slime mold, Dictyostelium discoideum, have been described. Many of these are regulated by cAMP. Analysis of the earliest time at which elevated levels of cAMP can induce the expression of these mRNAs reveals a more complex pattern of regulation in which genes change in their ability to be induced in response to cAMP with developmental stage. A prestalk mRNA (C1/D11) previously thought not be regulated by elevated levels of cAMP is inducible by cAMP between aggregation and loose mound stage; later in development its expression becomes independent of elevated cAMP. The early prespore genes (prespore class I) also show two modes of regulation; early in development they are induced independently of continuous elevated levels of cAMP, while later in development their expression is dependent upon elevated cAMP. The period during development when the prestalk genes are cAMP inducible precedes by 2 hr the first time at which either the early prespore class I or late prespore class II mRNAs are inducible by continuous elevated levels of cAMP. Previous analysis of these mRNAs has been carried out using Dictyostelium cells grown axenically. In this report we have studied the developmental expression of these mRNAs in cells grown on bacteria. A substantial shutoff of the class I prestalk and early prespore (class I) mRNAs not seen in axenically grown cells is observed when bacterially grown cells are plated for development. Less than 10% of the maximal level of these mRNAs remains in the cells at the time of mature spore and stalk differentiation. Additionally, in the bacterially grown cells two distinct patterns of developmental regulation are observed for mRNAs which in axenically growing cells appear to be constitutively expressed throughout growth and development.

Cyclic AMP and NH3/NH4+ both regulate cell-type-specific mRNA accumulation in the cellular slime mold, Dictyostelium discoideum.

Dictyostelium discoideum cells plated for development until aggregation stage, and then dissociated into media containing glucose, albumin, and cAMP will form into clumps and undergo prespore and prestalk differentiation. Differentiation in this in vitro system is dependent on three components: cAMP, multicellularity, and the acquisition of "differentiation competence" which the cells acquire in a period between interphase and aggregation stage when plated on Millipore filters. We have used this system to explore aspects of the multicellular environment which are involved in regulation the accumulation of the different prespore- and prestalk-specific messenger RNAs. Two classes of prespore messenger RNA, as well as a prestalk-specific messenger RNA, all require the acquisition of differentiation competence in order to be expressed in response to cAMP. Additionally, all of these messenger RNAs require agglomerate formation for maximal expression. The addition of 33 mM ammonium sulfate (NH4)2SO4, however, can entirely replace the requirement for agglomerate formation for expression of the prestalk-specific messenger RNA, and can partially substitute for agglomerate formation in inducing the expression of both classes of prespore-specific messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs while agglomerate formation or elevated NH3/NH+4 is essential only for the maintenance of the elevated levels of the messenger RNAs.

Interaction of cAMP with the cell-surface receptor induces cell-type-specific mRNA accumulation in Dictyostelium discoideum.

The accumulation of many postaggregative mRNA species in Dictyostelium discoideum is dependent upon the continuous presence of elevated levels of cAMP. We have analyzed the cyclic nucleotide specificity of this requirement and show that it is similar to that of the cell-surface receptor and distinct from the specificity displayed by the cAMP-dependent protein kinase. The same specificity is displayed for the accumulation of two classes of prespore mRNAs (class I, early; class II, late) and a prestalk mRNA and for the shutoff of a growth-phase mRNA. Under conditions in which cAMP phosphodiesterase activity is competitively inhibited, half-maximal accumulation of prestalk mRNA can be obtained at cAMP concentrations of 320-520 nM, whereas a higher concentration, 1-2 microM, is required for half-maximal accumulation of the prespore mRNAs and shutoff of the growth-phase mRNA. These effects of cAMP and its analogues on gene expression have been obtained under conditions in which cAMP-mediated activation of adenylate cyclase is completely inhibited. We conclude that cAMP acts to stimulate postaggregative gene expression by interacting at the cell-surface receptor.

Specific cell-cell contacts are essential for induction of gene expression during differentiation of Dictyostelium discoideum.

Postaggregation Dictyostelium discoideum cells contain 2000-3000 mRNA species that are absent from pre-aggregation cells. These aggregation-dependent sequences compose 30% of the mass of the late mRNA and represent the transcription products of an additional 11% of the single-copy genome. By analysis of mutants that are blocked at different stages of differentiation, we show that induction of expression of these genes is correlated with the formation of tight cell-cell contacts that resist EDTA. In particular, mutants that exhibit chemotaxis and aggregate to form loose mounds but do not form cell-cell contacts that resist EDTA fail to induce these late mRNA and protein species. By contrast, mutants that form normal contacts but progress no further through development do express the late mRNA species. Thus, interactions at the cell surface are involved in developmental induction of a large group of coregulated mRNAs. We have employed two independent assays for these developmentally regulated mRNAs: hybridization of gel-separated RNAs to cloned nuclear DNAs and hybridization of mRNA to a cDNA probe specific for the population of 2000-3000 regulated sequences.

Synthesis and stability of developmentally regulated dictyostelium mRNAs are affected by cell--cell contact and cAMP.

Postaggregation Dictyostelium discoideum cells contain 3000 mRNA species that are absent from preaggregation cells; these aggregation-dependent sequences comprise 30% of the mass of mRNA in these cells. We show that the synthesis and stability of these regulated mRNA sequences are affected by both cell--cell contact and cAMP. Three independent assays are used to quantitate these mRNAs: in vitro translation followed by two-dimensional gel analysis of the protein products; hybridization of gel-separated RNAs to cloned DNAs; and hybridization of mRNA to a cDNA probe specific for the population of regulated sequences. In postaggregation cells, the half-life of both the developmentally regulated mRNAs and the constitutive mRNAs present throughout growth and differentiation is the same--about 4 hr. Following disaggregation, all of the late mRNA sequences are degraded and decay with a half-life of 25 to 45 min. The constitutive species are unaffected; 2.5 hr after disaggregation, the ratio of late to constitutive mRNAs is about 6% that of normal plated cells. Addition of cAMP to cells that have been disaggregated for 2.5 hr (or longer) restores the level of most late mRNAs within 3 hr. We conclude that cAMP stimulates the synthesis of these mRNAs and may also act to stabilize them in the cytoplasm. This effect of cAMP is dependent on the cells having been in contact with other cells; cAMP has no effect on the levels of mRNA in suspension-starved, aggregation-competent cells that have never formed cell--cell aggregates.