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Query: UMLS:C0040822 (tremor)
 18,428 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have shown previously that expression of the Dictyostelium ras gene DdrasD (previously denoted Ddras) is induced during multicellular development and in single-cell shaking culture in response to cAMP (1). Analysis of transformants carrying DdrasD/lacZ reporter constructs showed DdrasD expression to be prestalk-specific (2). The gene is transcribed from three start sites with transcription from the distal site producing an approximately 1.2 kb transcript, which is expressed at low levels in growing cells and is subsequently induced late in aggregation. This promoter is also induced to high levels by cAMP. Transcription from the two more proximal sites is coregulated and is induced during development, resulting in approximately 1.0 kb transcripts. In this study, we examine cis-acting regions required for proper regulation of DdrasD expression using a DdrasD/beta-glucuronidase reporter gene construct. We have identified distinct sequence elements required for developmental and vegetative expression of DdrasD. A domain containing a CA repeat, similar to ones found in other late, cAMP-induced Dictyostelium genes, is required for cAMP-induced and developmental expression.
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PMID:Regulation of the Dictyostelium cAMP-induced, prestalk-specific DdrasD gene: identification of cis-acting elements. 131 67

DdrasG gene expression during the early development of Dictyostelium discoideum has been examined in detail. The amount of DdrasG-specific mRNA increased approximately twofold during the first 2 to 3 h of development and then declined rapidly, reaching negligible levels by the aggregation stage. The increase in mRNA levels that occurred during the first 2 to 3 h of development also occurred during differentiation in cell suspensions and was enhanced when cells were shaken rapidly. This initial increase was unaffected by cell density. When cells were set up to differentiate on filters, the addition of a glucose-amino acid mixture slightly delayed differentiation and had a similar effect on the expression of the gene. The decline in DdrasG expression during development did not occur when cells were treated with cycloheximide, suggesting that the expression of a developmentally regulated gene product is essential for the reduction of DdrasG gene mRNA. There was no decrease in DdrasG mRNA level during differentiation in shake suspension, but the decrease did occur upon application of pulses of cyclic AMP to shaking cultures. The application of a continuously high level of cyclic AMP delayed the increase in expression of the gene and did not result in the subsequent decline. These results suggest that the induction of a functional cyclic AMP relay system is important in reducing DdrasG gene mRNA levels.
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PMID:Regulation of DdrasG gene expression during Dictyostelium development. 215 84

Previously, we identified a class of genes in Dictyostelium that are prespore cell-type specific in their expression in the multicellular aggregate and are inducible by cAMP acting through cell-surface cAMP receptors. In this paper, we report the cloning and analysis of the regulatory regions controlling the expression of one such gene that encodes a spore coat protein, SP60. By use of a fusion of the firefly luciferase gene and Escherichia coli lacZ [expresses beta-galactosidase (beta-gal)], we have identified cis-acting regions required for proper spatial and temporal expression in multicellular aggregates and for cAMP induction in shaking cell culture. Deletion analysis suggests that a CA-rich element (CAE) and surrounding sequences present three times within the 5'-flanking sequence are required for proper regulation. SP60-lacZ fusions that include all three of these regions express lacZ only in the posterior approximately 85% of migrating slugs (prespore zone). Studies show that SP60 is expressed during mid to late aggregation, and SP60-lacZ-positive cells are spatially localized as a doughnut-shaped ring within the forming aggregate. Cells within the skirt that surrounds the aggregate and that are still migrating into the aggregate do not stain. Sequential 5' deletions of CAEs and surrounding regions affect the expression level of SP60-luciferase in response to developmental signals and cAMP, as well as the spatial pattern of SP60-lacZ. Deletion of the first (most 5') of these regions restricts the spatial expression of SP60-lacZ fusions to the anterior of the prespore zone. When both the first and second regions are removed, the expression level drops, and the staining is restricted to the prespore/prestalk boundary. Furthermore, the staining pattern that is seen with these two deletions is present as a gradient from anterior to posterior within the prespore zone. Deletion of all three regions results in a loss of both cAMP and developmentally induced expression. These results suggest the presence of a gradient within the prespore zone that differentially affects the activity of promoters containing different numbers of response elements.
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PMID:A spatial gradient of expression of a cAMP-regulated prespore cell-type-specific gene in Dictyostelium. 216 44

We have recently reported the existence of two forms of glycogen phosphorylase (1,4-alpha-D-glucan: orthophosphate-alpha-glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5'AMP and a 104 kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 microM, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt2-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 microM) for 'b' to 'a' conversion. cDNAs to three cAMP-regulated genes--PL3, D11, and D3--were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture--a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.
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PMID:Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum. 217 98

We have previously shown that several genes expressed during Dictyostelium development could be induced in shaking culture by exogenous cAMP, even though the accumulation of intracellular cAMP was inhibited. The use of selected cAMP analogs indicated that the exogenous cAMP functioned by activating the cell surface cAMP receptor and not by interacting with the regulatory subunit of the intracellular cAMP-dependent protein kinase. Although some genes in Dictyostelium appear to be regulated by intracellular cAMP, these data suggest that this is not the case for all genes regulated by cAMP. Intracellular second messengers other than cAMP may, therefore, promote the expression of these other genes. Here, we have examined inositol trisphosphate and diacylglycerol as candidates for such mediators of signal transduction. We have studied three genes that exhibit disparate modes of temporal and spatial expression during development of Dictyostelium. In shaking cultures, maximal levels of expression of each are dependent on the accumulation of or exposure to extracellular cAMP. We show that the addition of inositol trisphosphate and/or diacylglycerol to cells in shaking culture has distinct effects on the expression of each gene and, under specific conditions, can bypass the requirement for extracellular cAMP. These data suggest that extracellular cAMP interacting with its cell surface receptor may promote synthesis of inositol trisphosphate and diacylglycerol to regulate gene expression and aspects of differentiation in Dictyostelium.
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PMID:Inositol trisphosphate and diacylglycerol can differentially modulate gene expression in Dictyostelium. 255 9

By the use of an in vivo assay, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) is shown to be developmentally regulated in Dictyostelium discoideum. High levels of cAMP can induce ornithine decarboxylase activity in preaggregative cells kept in shaking suspension, under similar conditions as where other markers for development can also be induced. This induction by cAMP is solely dependent on the total amount of cAMP to which the cells have been exposed, and not on the manner of cAMP addition. Induction of ornithine decarboxylase activity, when measured in vitro, is caused by both an increase in total enzyme activity and by a proportional increase in activity of the high-affinity form for the cofactor pyridoxal phosphate. When measured in vivo, an additional regulatory mechanism seems to be involved. Kinetic studies with the competitive inhibitor putrescine suggest that in cAMP-stimulated cells the low affinity form of the enzyme may also be active in vivo.
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PMID:The developmental regulation of L-ornithine decarboxylase in Dictyostelium discoideum and its induction by cAMP. 299 May 80

We describe the gene M4-1, whose unique pattern of developmental expression will allow us to study the molecular mechanisms controlling expression in undifferentiated cells in addition to repression in response to cAMP during development and reinduction during dedifferentiation. M4-1 is a Dictyostelium gene expressed in the undifferentiated cell. We have shown that M4-1 continues to be expressed very early during the developmental cycle but is repressed at a later stage of development, at a time coincident with the establishment of oscillations in the cAMP pool. Studies on the expression of the M4-1 gene in shaking culture, under conditions that mimic early development, have established that pulsatile stimulation of cells with cAMP is sufficient to repress M4-1 expression. Consistent with this, cells that are exposed to high levels of cAMP are unable to respond normally to cAMP oscillations and continue to express M4-1 at vegetative levels. These data indicate that low-level oscillations of cAMP are required for the repression of M4-1 expression rather than the continuous high levels of cAMP responsible for the regulation of a different class of Dictyostelium genes. We suggest that cAMP may mediate developmental expression of the Dictyostelium genome by different mechanisms. We also show that cell-cell interaction, a developmental event that occurs subsequent to the cAMP pulse, does not normally influence the regulation of M4-1. Finally, we have shown that when cAMP-pulsed cells are induced to dedifferentiate, M4-1 RNA sequences rapidly reappear in nuclei and cytoplasm, suggesting that regulation of M4-1 expression is primarily mediated at the level of transcription.
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PMID:A gene expressed in undifferentiated vegetative Dictyostelium is repressed by developmental pulses of cAMP and reinduced during dedifferentiation. 301 Mar 12

Extracellular adenosine 3',5'-cyclic monophosphate (cAMP) is required for cell-type-specific gene expression in developing Dictyostelium discoideum. We have developed a microassay for the expression of these genes, using antibodies directed against their protein products. To characterize the transduction mechanism, we have used in this assay cAMP analogues that preferentially activate either the cell-surface cAMP receptor or the internal cAMP-dependent protein kinase. N6-(aminohexyl) cAMP activates the Dictyostelium cAMP-dependent protein kinase but does not bind to the cell-surface cAMP receptor and does not cause cell-type-specific gene expression. 2'-Deoxy-cAMP does not activate the cAMP-dependent protein kinase but binds to the receptor and causes cell-type-specific gene expression. Cyclic AMP-induced accumulation of prestalk mRNA in shaking cultures still occurs in the presence of caffeine, which blocks the receptor-coupled activation of adenyl cyclase. This suggests that the extracellular cAMP induction of cell-type-specific gene expression in developing Dictyostelium cells is mediated by the cell-surface cAMP receptor and that activating adenyl cyclase by this receptor is not essential. Using the N6-(aminohexyl) cAMP to competitively inhibit phosphodiesterase, we show that 30 nM cAMP is sufficient to induce prestalk or prespore gene expression.
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PMID:cAMP induction of prespore and prestalk gene expression in Dictyostelium is mediated by the cell-surface cAMP receptor. 302 99

We examined two sets of genes expressed early in the developmental cycle of Dictyostelium discoideum that appear to be regulated by cyclic AMP (cAMP). The transcripts of both sets of genes were not detectable in vegetative cells. During normal development on filter pads, RNA complementary to these genes could be detected at about 2 h, peaked around 6 to 8 h, and decreased gradually thereafter. Expression of these genes upon starvation in shaking culture was stimulated by pulsing the cells with nanomolar levels of cAMP, a condition that mimics the in vivo pulsing during normal aggregation. Expression was inhibited by caffeine or by continuous levels of cAMP, a condition found later in development when in vivo expression of these genes decreased. The inhibition of caffeine could be overcome by pulsing cells with cAMP. These results suggest that the expression is mediated via the cell surface cAMP receptor, but does not require a rise in intracellular cAMP. mRNA from a gene of the second class was induced upon starvation, peaked by 2.5 h of development, and then declined. In contrast to the other genes, its expression was maintained by continuous levels of cAMP and repressed by cAMP pulses. These and other results on a number of classes of developmentally regulated genes indicates that changing levels of cAMP, acting via the cell surface cAMP receptor, are involved in controlling these groups of genes. We also examined the structure and partial sequence of the cAMP pulse-induced genes. The two tandemly duplicated M3 genes were almost continuously homologous over the sequenced portion of the protein-coding region except for a region near the N-terminal end. The two M3 genes had regions of homology in the 5' flanking sequence and showed slight homology to the same regions in gene D2, another cAMP pulse-induced gene. D2 showed extremely significant homology over its entire sequenced length to an acetylcholinesterase. The results presented here and by others suggest that expression of many early genes in D. discoideum is regulated via the cell surface cAMP receptor. We expect that many of these genes may play essential roles in early Dictyostelium development and could code for elements of the cAMP signal transduction pathway involved in aggregation.
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PMID:Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor. 303 75

Constructs were made in which approximately equal to 500 base pairs of the 5' flanking region of the ras gene of Dictyostelium discoideum and variable amounts of the coding region were linked to a ras cDNA in a transformation vector. These constructs then were used to transform Dictyostelium cells and their regulation was examined. In Dictyostelium transformants, transcripts from the ras gene constructs were found at high levels in cells in fast-shaking cultures containing cAMP, whereas transcripts were either not detectable or present at very low levels in cultures lacking exogenous cAMP. In slow-shaking culture, a significantly lower level of ras RNA was detected. When normal developing aggregates were dissociated, RNA from the ras constructs decreased rapidly but then reaccumulated in the presence of cAMP. These results show that the sequences necessary for the response to external cAMP are present within an approximately equal to 650-base-pair region upstream from the ATG start codon and/or within portions of the protein-coding region. Moreover, the proper regulation of ras gene expression in high-copy-number transformants suggests that trans-acting factors which may control transcription are not limiting. Vector constructs were also examined in which the cDNA was present in the opposite orientation compared to the gene fragment (antisense orientation). When these were transfected into cells, no transformants were obtained, suggesting that expression of the ras gene is essential for vegetative growth.
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PMID:Regulated expression of ras gene constructs in Dictyostelium transformants. 386 37


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