Altered morphology of vegetative amoebae induced by increased expression of the Dictyostelium discoideum ras-related gene rap1

The rap1 gene of Dictyostelium discoideum is a member of the ras-gene superfamily of low molecular weight GTPase proteins. The rapl gene is expressed both during growth and development in D. discoideum. To examine the action of the Rapl protein in D. discoideum, the rap1 cDNA was expressed under the control of the inducible discoidin promoter. Treatment with conditioned media, which induces the discoidin promoter, increased Rap1 protein levels in vegetative cells approximately six fold. Overexpression of the Rapl protein correlated with the appearance of morphologically aberrant vegetative amoebae: cells were extensively spread and flattened. The distribution of F-actin was altered in these cells, with an increase in actin staining around the cell periphery. Induction of the discoidin promoter by starvation in the rapl transformants also resulted in spread flat cells. When starved D. discoideum amoebae are refed with HL5 media, the cells rapidly respond by rounding up. By contrast, the rapl transformant cells showed a pronounced delay in rounding up. Rapid tyrosine phosphorylation of a p45 protein occurred in both control cells and the rapl transformant upon refeeding, implying that the signal transduction pathway leading to tyrosine phosphorylation remained functional in the rapl transformant. We propose that the Rapl protein functions in the regulation of cell morphology in D. discoideum. © 1993Wiley-Liss, Inc.     

Specific expression of a gene encoding a novel calcium-binding protein, CAF-1, during transition of Dictyostelium cells from growth to differentiation

The gene expressions involved in the transition from cell proliferation to differentiation were analyzed, using synchronized Dictyostelium discoideum Ax-2 cells and the differential plaque hybridization method. As one of the genes (cDNA) specifically expressed when Ax-2 cells were starved just before the putative shift (PS)-point (putative shift point; a switchover point from growth to differentiation in the cell cycle), calfumirin-1 ( CAF-1 ) was cloned, which encoded a novel calcium-binding protein with E-F hand. Although CAF-1 mRNA was slightly expressed in vegetatively growing cells, the expression was markedly increased in response to starvation of cells just before the PS-point. Northern analysis using non-synchronized Ax-2 cells showed that the CAF-1 mRNA is predominantly expressed within a few hours of starvation. Such a starvation-induced early expression of the CAF-1 mRNA raised a possibility that CAF-1 might be one of Ca²⁺-binding proteins involved in the phase-shift of cells from growth to differentiation.     

A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior-like cells during development of Dictyostelium

The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell-substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicates that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage, however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup, and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk.     

Regulation of discoidin I gene expression in dictyostelium discoideum by cell-cell contact and cAMP

We have previously presented evidence that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression in D discoideum [Berger EA, Clark JM: Proc Natl Acad Sci USA 80:4983, 1983]. Here we provide genetic evidence to support this hypothesis by examining gene expression in a cohesion-defective mutant, strain EB-21, which enters the developmental program but is blocked at the loose mound stage. When this strain was developed in suspension, the cells remained almost entirely as single amoebae, unlike the wild type, which formed large multicellular aggregates. In both strains, discoidin I mRNA levels were low in vegetative cells but rose sharply during the first few hours of development. However, the peak level reached at 8 hr in EB-21 exceeded that observed in wild type, and while the level declined markedly over the next few hours in wild type, it remained highly elevated in the mutant. Thus, there was a correlation between the inability of EB-21 to form normal cell-cell contacts and its deficiency in inactivating discoidin I gene expression. Previous studies from several laboratories, including this one, have demonstrated that exogenously added cAMP can block or reverse the changes in gene expression normally seen upon cell disaggregation. This has led us to propose that cAMP serves as a second messenger regulating the expression of contact-regulated genes. Here we provide additional support for this hypothesis. Intracellular cAMP levels rapidly dropped several-fold when wild type tight cell aggregates were disaggregated and remained low as the cells were cultured in the disaggregated state. Furthermore, overexpression of discoidin I mRNA late in development in EB-21 was corrected by addition of high concentrations of cAMP. These results are consistent with a second messenger function for cAMP in the contact-mediated regulatory response, and they indicate that the cAMP response machinery for discoidin I gene expression is capable of functioning in the cohesion-defective EB-21 strain.     

Identification of transposable elements which activate gene expression in Pseudomonas cepacia.

This study demonstrated that transposable elements in Pseudomonas cepacia could be inserted upstream of a poorly expressed gene and increase its expression more than 30-fold. Five elements, TnPc1, IS402, IS403, IS404, and IS405, were isolated by their ability to increase expression of the beta-lactamase gene of the broad-host-range plasmid pRP1. Increased expression resulted only from insertion of these elements, suggesting that insertional activation is an important means of elevating gene expression in this organism. Four of the elements inserted between a PstI site within the beta-lactamase gene and a BamHI site located 375 base pairs upstream of its promoter. The element IS403 inserted distal to the BamHI site within the coding region for the gene tnpR, suggesting that insertional activation can act over greater than expected distances. In addition, the element IS402 activated the beta-lactamase genes carried on plasmids pRP1 and pMR5 (temperature-sensitive pRP1) equally well in opposite orientations, demonstrating that insertional activation by this element occurs independent of its orientation.     

Enhancers predominantly regulate gene expression during differentiation via transcription initiation

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Temporal and spatial expression of ammonium transporter genes during growth and development of Dictyostelium discoideum

Ammonia is an important signaling molecule involved in the regulation of development in Dictyostelium. During aggregation, ammonia gradients are established, and the ammonia concentration in the immediate environment or within a particular cell throughout development may vary. This is due to the rate of cellular ammonia production, its rate of loss by evaporation to the atmosphere or by diffusion into the substratum, and perhaps to cellular transport by ammonium transporters (AMTs). Recent efforts in genome and cDNA sequencing have identified three ammonium transporters in Dictyostelium. In addition to physically altering the levels of ammonia within cells, AMTs also may play a role in ammonia signaling. As an initial step in identifying such a function, the temporal and spatial expression of the three amt genes is examined. RT-PCR demonstrates that each of the three amt mRNAs is present and relatively constant throughout growth and development. The spatial expression of these three amt genes is examined during multiple stages of Dictyostelium development using in situ hybridization. A distinct and dynamic pattern of expression is seen for the three genes. In general, amtA is expressed heavily in pre-stalk cells in a dynamic way, while amtB and amtC are expressed in pre-spore regions consistently throughout development. AmtC also is expressed in the most anterior tip of fingers and slugs, corresponding to cells that mediate ammonia's effect on the choice between slug migration and culmination. Indeed, amtC null cells have a slugger phenotype, suggesting AmtC functions in the signaling pathway underlying the mechanics of this choice.     

Non-LTR retrotransposons with unique integration preferences downstream of Dictyostelium discoideum tRNA genes

Retrotransposable elements are genetic entities which move and replicate within host cell genomes. We have previously reported on the structures and genomic distributions of two non-long terminal repeat (non-LTR) retrotransposons, DRE and Tdd-3, in the eukaryotic microorganism Dictyostelium discoideum. DRE elements are found inserted upstream, and Tdd-3 elements downstream, of transfer RNA (tRNA) genes with remarkable position and orientation specificities. The data set currently available from the Dictyostelium Genome Project led to the characterisation of two repetitive DNA elements which are related to the D. discoideum non-LTR retrotransposon Tdd-3 in both their structural properties and genomic distributions. It appears from our data that in the D. discoideum genome tRNA genes are major targets for the insertion of mobilised non-LTR retrotransposons. This may be interpreted as the consequence of a process of coevolution, allowing a viable population of retroelements to transpose without being deleterious to the small microbial host genome which carries only short intergenic DNA sequences. A new nomenclature is introduced to designate all tRNA gene-targeted non-LTR retrotransposons (TREs) in the D. discoideum genome. TREs inserted 5′ and 3′ of tRNA genes are named TRE5 and TRE3, respectively. According to this nomenclature DRE and Tdd-3 are renamed TRE5-A and TRE3-A, respectively. The new retroelements described in this study are named TRE3-B (formerly RED) and TRE3-C. Received: 27 May 1999 / Accepted: 23 July 1999