Chromatin organisation of transgenes in Dictyostelium.

The introduction of transgenes in Dictyostelium discoideum typically results in the integration of the transformation vector into the genome at one or a few insertion sites as tandem arrays of approximately 100 copies. Exceptions are extrachromosomal vectors, which do not integrate into chromosomes, and vectors containing resistance markers such as blasticidin, which integrate as single copies at one or a few sites. Here we report that low copy number vector inserts display typical euchromatic features while high copy number insertions are enriched for modifications associate with heterochromatin. Interestingly, high copy number insertions also colocalise with heterochromatin, are enriched for the centromeric histone CenH3 and display centromere-like behaviour during mitosis. We also found that the chromatin organisation on extrachromosmal transgenes is different from those integrated into the chromosomes.

Binding of IRE-BP to its cognate RNA sequence: SFM studies on a universal RNA backbone for the analysis of RNA-protein interaction.

We have used an RNA consisting of the potato spindle tuber viroid (PSTVd) and 240 bp of double-stranded RNA derived from the GUS gene as a backbone for scanning force microscope (SFM) studies on RNA binding proteins. The in vitro transcribed RNA forms a rod-like structure of apparent 130 nm in length with a completely base paired central part flanked by the incompletely paired viroid helix with bulges on both sides. The termini of the molecule consist of loops such that no blunt or staggered RNA ends are exposed. Suitable, asymmetrical restriction sites in the construct allow for the insertion of sequences of interest, e. g. protein binding sites. We have inserted the IRE (iron responsive element) sequence into the construct and have used in vitro transcripts to study binding of IRE-BP. Relative binding frequencies show that 70% of the protein binds to the expected site in the molecule while only a slightly enhanced binding is observed at the termini. In the GUS-PSTVd-IRE backbone, the orientation of the molecule is easily determined by IRE-BP binding. It thus provides a versatile tool to study specific as well as preferential interaction of other proteins with sequences or structures inserted into a different part of the molecule.

Interaction of gdt1 and protein kinase A (PKA) in the growth-differentiation-transition in Dictyostelium.

The gdt1 gene is a negative regulator of the growth-differentiation-transition (GDT) in Dictyostelium. gdt1- cells express the GDT marker discoidin earlier and at higher levels and prematurely enter the differentiation pathway. Protein kinase A is a positive regulator of the GDT and is required for multicellular development. Disruption of the PKA catalytic subunit or overexpression of a constitutively active mutant of the regulatory subunit results in cells which do not form multicellular aggregates and which show strongly reduced levels of discoidin. We have created PKA-/gdt1- double mutants and show that these display high levels of discoidin expression but no aggregation, suggesting that gdt1 may be a downstream target of PKA in a branched signaling cascade initiating differentiation. Data obtained with the PKA inhibitor H89 support these result: in wild type cells H89 inhibits discoidin expression while in gdt1- mutants there is no obvious effect. However, since PKA-/gdt1- cells display less discoidin expression than the single gdt1 mutant, we propose that PKA and gdt1 are in two parallel interacting pathways. To get insight into the mechanism how PKA may block gdt1, we have tested two putative PKA phosphorylation sites in the protein and found that one of them is efficiently phosphorylated by PKA in vitro. A model for the interplay between PKA and gdt1 during the growth-differentiation-transition is discussed.

gdt1, a new signal transduction component for negative regulation of the growth-differentiation transition in Dictyostelium discoideum.

Discoidin I expression was used as a marker to screen for mutants affected in the growth-differentiation transition (GDT) of Dictyostelium. By REMI mutagenesis we have isolated mutant 2-9, an overexpressor of discoidin I. It displays normal morphogenesis but shows premature entry into the developmental cycle. The disrupted gene was denominated gdt1. The mutant phenotype was reconstructed by disruptions in different parts of the gene, suggesting that all had a complete loss of function. gdt1 was expressed in growing cells; the levels of protein and mRNA appear to increase with cell density and rapidly decrease with the onset of development. gdt1 encodes a 175-kDa protein with four putative transmembrane domains. In the C terminus, the derived amino acid sequence displays some similarity to the catalytic domain of protein kinases. Mixing experiments demonstrate that the gdt1(-) phenotype is cell autonomous. Prestarvation factor is secreted at wild-type levels. The response to folate, a negative regulator of discoidin expression, was not impaired in gdt1 mutants. Cells that lack the G protein alpha2 display a loss of discoidin expression and do not aggregate. gdt1(-)/Galpha2(-) double mutants show no aggregation but strong discoidin expression. This suggests that gdt1 is a negative regulator of the GDT downstream of or in a parallel pathway to Galpha2.

Role of cAMP-dependent protein kinase during growth and early development of Dictyostelium discoideum.

cAMP-dependent protein kinase (PKA) is an essential regulator of gene expression and cell differentiation during multicellular development of Dictyostelium discoideum. Here we show that PKA activity also regulates gene expression during the growth phase and at the transition from growth to development. Overexpression of PKA leads to overexpression of the discoidinIgamma promoter, while expression of the discoidinIgamma promoter is reduced when PKA activity is reduced, either by expression of a dominant negative mutant of the regulatory subunit or by disruption of the gene for the catalytic subunit (PKA-C). The discoidin phenotype of PKA-C null cells is cell autonomous. In particular, normal secretion of discoidin-inducing factors was demonstrated. In addition, PKA-C null cells are able to respond to media conditioned by PSF and CMF. We conclude that PKA is a major activator of discoidin expression. However, it is not required for production or transduction of the inducing extracellular signals. Therefore, PKA-dependent and PKA-independent pathways regulate the expression of the discoidin genes.

Determination of preferential binding sites for anti-dsRNA antibodies on double-stranded RNA by scanning force microscopy.

The monoclonal anti-dsRNA antibody J2 binds double-stranded RNAs (dsRNA) in an apparently sequence-nonspecific way. The mAb only recognizes antigens with double-stranded regions of at least 40 bp and its affinity to poly(A) poly(U) and to dsRNAs with mixed base pair composition is about tenfold higher than to poly(I) poly(C). Because no specific binding site could be determined, the number, the exact dimensions, and other distinct features of the binding sites on a given antigen are difficult to evaluate by biochemical methods. We therefore employed scanning force microscopy (SFM) as a method to analyze antibody-dsRNA interaction and protein-RNA binding in general. Several in vitro-synthesized dsRNA substrates, generated from the Dictyostelium PSV-A gene, were used. In addition to the expected sequence-nonspecific binding, imaging of the complexes indicated preferential binding of antibodies to the ends of dsRNA molecules as well as to certain internal sites. Analysis of 2,000 bound antibodies suggested that the consensus sequence of a preferential internal binding site is A2N9A3N9A2, thus presenting A residues on one face of the helix. The site was verified by site-directed mutagenesis, which abolished preferential binding to this region. The data demonstrate that SFM can be efficiently used to identify and characterize binding sites for proteins with no or incomplete sequence specificity. This is especially the case for many proteins involved in RNA metabolism.

Production and activity of spore differentiation factors (SDFs) in Dictyostelium.

SDF-1 and SDF-2 are peptides that promote terminal spore differentiation under submerged conditions. The present study shows that they accumulate differentially and are released during the development of wild-type cells and can promote spore formation in cells disaggregated from wild-type culminants. SDF-1 accumulates during the slug stage and is released in a single burst at the onset of culmination while SDF-2 accumulates during early culmination and is released in a single burst from mid-culminants. The effects of SDF-1 and SDF-2 on stalk cell formation in cell monolayers were investigated. SDF-1 by itself induces stalk cell formation in some strains and also synergizes with the stalk-cell-inducing factor, DIF-1. cAMP has an inhibitory effect on stalk cell formation when either DIF-1 or SDF-1 are present on their own but is almost not inhibitory when both are present. SDF-2 alone does not induce stalk cell formation and appears to inhibit the response to DIF-1. At the same time, it increases the extent of vacuolization of the stalk cells that are produced. We propose that the release of SDF-1 and then of SDF-2 may mark irreversible steps in the developmental programme associated, respectively, with culmination and spore maturation.

Signal transduction pathways leading to spore differentiation in Dictyostelium discoideum.

Cells that overexpress PKA as a consequence of carrying multiple copies of the gene encoding the catalytic subunit can be induced to sporulate when developing as single cells. A peptide phosphorylated by PKA, termed SDF-1, has recently been shown to stimulate this process (Anjard et al., 1997). Several genes have been implicated in a signal transduction pathway by which prestalk cells induce encapsulation of prespore cells during terminal differentiation including a prestalk-specific putative membrane protease (TagC) and a two-component system consisting of a receptor-histidine kinase (DhkA) and a response regulator with cAMP phosphodiesterase activity (RegA). To determine whether SDF-1 uses this pathway, strains carrying null mutations in the pertinent genes were transformed with a pkaC plasmid such that they can overexpress PKA. Since these mutant strains all sporulated efficiently when SDF-1 was added, it appears that other gene products mediate the response. However, we found that regA- mutant cells release a distinct factor, SDF-2, that rapidly induces encapsulation of test cells overexpressing pkaC. Since cells in which tagC is disrupted do not form SDF-2 and cells in which dhkA is disrupted do not respond to SDF-2, this peptide appears to use the two-component system that regulates PKA activity. SDF-2 is a small peptide released by prestalk cells in a manner dependent on TagC. It appears to act on prespore cells through the DhkA receptor to inhibit the cAMP phosphodiesterase of RegA, thereby activating PKA via cAMP. The process of induction by SDF-2 can be shown to be distinct from that by SDF-1. SDF-2 appears to stimulate prestalk cells to release additional SDF-2 by acting through a signal transduction pathway that also involves DhkA, RegA, and PKA. Based on these results we present a model for the signal transduction cascade regulating spore differentiation.

Functional activity and developmental regulation of DdRBP1, a RNA binding protein in Dictyostelium discoideum.

In an attempt to find potential components of natural antisense mechanisms in Dictyostelium, we investigated RNA binding protein (RBD) genes of the RNP-CS family. RBD proteins can enhance hybridization of complementary RNAs and may thus mediate the interaction of sense and antisense RNA. Using the conserved RNP1 and RNP2 motifs as primers, we cloned 4 PCR fragments containing ORFs and additional homologies to known members of the RNP-CS family. We cloned a full length cDNA for one protein (DdRBP1) that showed similarities to hnRNP A1. Recombinant protein synthesized in E. coli displayed binding to single stranded RNA and a weak annealing activity for partially complementary RNAs in vitro. Deletion of the RNP1 motif reduced RNA binding considerably but not completely. DdRBP1 is thus one of the few members of the RNP-CS family for which binding and annealing activities have been experimentally demonstrated. Polyclonal antisera directed against recombinant DdRBP1 detected a protein of approx. 40 kDa. In whole cell extracts, this protein was present in equal amounts throughout the developmental cycle of Dictyostelium while differential accumulation was observed in nuclei during early and late development.

In vitro and in vivo action of antisense RNA.

The transient or permanent expression of antisense RNA represents one option to apply antisense techniques in biotechnology and medical research. Despite the increasing importance and use of antisense nucleic acids as well as their significant antisense-specific phenotypic effects in vivo, there is an obvious lack of explanation for the mechanism of their action. By studying naturally occurring antisense RNA and analyzing their mechanism of action we attempt to learn more about the design, the use, and the critical parameters of artificial antisense RNA. Attempts to derive models from biochemical and structural studies for the interactions between antisense RNAs and their targets will be discussed.

Antagonistic effects of signal transduction by intracellular and extracellular cAMP on gene regulation in Dictyostelium.

In Dictyostelium, cAMP plays a role as an intracellular second messenger and in addition, as an extracellular first messenger. Both functions are thought to be tightly linked because adenylyl cyclase is coupled via G-proteins to the cell surface cAMP receptor cAR 1. Using the discoidin I gene family as a molecular marker for the first stages of development, we show here that induction of transcription requires the G-protein subunit alpha 2 and thus an as yet unidentified surface receptor, CRAC (cytosolic regulator of adenylyl cyclase), and PKA. Induction can be conferred by an increase in intracellular cAMP. In contrast, transcriptional down-regulation occurs by stimulation of cAR 1 with extracellular cAMP and a subsequent, G-protein-independent Ca2+ influx. In a G alpha 2 gene disruption mutant, discoidin I expression can be efficiently modulated by analogues simulating intracellular cAMP (discoidin induction) and extracellular cAMP (discoidin down-regulation). We thus demonstrate possible antagonistic functions of intra- and extracellular cAMP.

Analysis of gene function in Dictyostelium.

Over the past ten years, powerful molecular genetic techniques have been developed to analyze gene function in Dictyostelium. DNA-mediated transformation using a variety of selections and vectors has allowed the introduction of wild-type or modified genes that are under various forms of transcriptional control. Homologous recombination is efficient and can be used to modify the genome in precise ways. In addition, it is now possible to clone genes based on their mutant phenotype alone, either by insertional mutagenesis, or by screening antisense expression cDNA libraries. Finally, a nearly complete physical map of the genome is available and so genes are easily mapped by physical techniques. We discuss many of these advances within the context of major research problems presently under study.

Multiple signal transduction pathways regulate discoidin I gene expression in Dictyostelium discoideum.

The expression of the discoidin I genes in Dictyostelium discoideum is regulated by the concerted action of the extracellular factors cyclic adenosine monophosphate (cAMP), folate, prestarvation factor (PSF) and conditioned media factor (CMF). However, the pathways by which these signals are transduced and the interactions between the pathways have been unexplored so far. We have analysed wild-type and mutant cells with defined lesions in signal transduction to elucidate these regulatory processes, and shown that different pathways are used for the down-regulation and induction of these genes. The cAMP receptor cARI is required for the cAMP-mediated down-regulation of discoidin I gene expression but not for the induction of discoidin I expression during development. Surprisingly, induction of the discoidin I genes requires G alpha 2, the G-protein subunit which is generally believed to couple to cARI, to control the expression of cAMP-inducible genes. Thus, our data suggest that G alpha 2 interacts with different receptors to regulate gene expression in early development. Furthermore, the analysis shows that discoidin induction in bacterially grown cells occurs in two sequential steps. The first is a low basal induction which occurs in late log-phase growth prior to starvation. PSF can induce the basal level, and the induction is independent of G alpha 2. The developmental induction following starvation is much stronger, dependent on G alpha 2 and probably signaled by CMF, which is secreted at that time.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental regulation of DEAD box proteins and cloning of putative RNA helicase genes from Dictyostelium discoideum.

RNA secondary structure is essential for RNA function in pre-mRNA splicing, mRNA translation, ribosome assembly and RNA stability. The involvement of DEAD/H RNA helicases in the regulation of these processes has been demonstrated in some cases. To investigate the repertoire of DEAD box proteins expressed in Dictyostelium discoideum, we used PCR techniques to clone two cDNAs coding for DEAD box proteins with high similarity to known yeast proteins: Dictyostelium Hel2A is about 45% identical to Saccharomyces cerevisiae DBP2 and S. pombe dbp2, the yeast homologues of human p68. Dictyostelium Hel2B is about 43% identical to the S. cerevisiae splicing factor PRP28, but has a different domain at the N-terminus, which is unique for Dictyostelium discoideum. Using a polyclonal antibody directed against a DEAD box peptide we show differential expression of three DEAD box proteins during the developmental cycle of Dictyostelium.

dsRNA degrading nucleases are differentially expressed in tobacco anthers.

Nucleases, capable of digesting double-stranded RNAs are mainly confined to extracellular fractions of tobacco anthers and diffusate of mature pollen. dsRNAse activity is about 150-fold higher in anther fractions than in crude nuclease extracts from tobacco leaves. The level of dsRNAse activity varies during pollen development from the microspore stage to maturity. In the anther soluble fraction, dsRNAse activity reached a maximum (approx. 50 units/anther) at the end of microspore mitosis and then decreased continuously until the stage of almost mature anthers. In contrast, the nuclease activity associated with pollen increased continuously reaching a maximum (5 units/anther), during subsequent stages of pollen maturation. Gel electrophoretic analysis revealed four slowly migrating sugar-unspecific nucleases (active against DNA and RNA) and three faster migrating RNases which were all able to digest dsRNA. Competition experiments showed that the sugar-unspecific nucleases accounted for 95% of the total dsRNAse activity. Anther extracellular nucleases were further characterized after partial purification on NADP-agarose: dsRNAse activity had a pH optimum at 5.5, was strongly inhibited by NaCl and by 1 mM Zn2+ and was insensitive to EDTA which could stimulate activity in crude preparations. Analysis of the activity with defined substrates showed that ssRNA is more readily degraded than dsRNA and that both, endo- and exonucleolytic activities are detected.

Developmental regulation of antisense-mediated gene silencing in Dictyostelium.

In Dictyostelium, the expression of antisense transcripts has been successfully used to reduce or eliminate gene expression. In most cases this occurs on the level of RNA stability resulting in a loss of both sense and antisense transcript accumulation. We here show that the antisense effect is regulated during the developmental cycle, i.e., in certain developmental stages and under certain developmental conditions, complementary RNAs appear not to interact with each other, resulting in a failure to abolish expression of the gene of interest. We find that this is not only the case with artificially introduced antisense constructs but also with the endogenous, antisense-regulated PSV-A gene. Our data demonstrate that antisense-mediated gene silencing is conferred by a biochemical machinery that is subject to regulation in vivo. The results provide a basis to better understand this machinery and to dissect the components. They may also explain the failure of some antisense experiments in Dictyostelium and possibly in other organisms.

Diversification of antisense research and development: review of the Ringberg meeting, April 1994. Mechanisms of antisense-mediated gene silencing.

Antisense technology has established itself as a new and vibrant entrant into the discipline of molecular biology. As such, it has contributed to basic research by providing tools for the molecular dissection of diverse experimental systems. In applied research, antisense approaches have contributed to development of agricultural products (D. Grierson) now coming to market and to the design of a number of oligonucleotide drugs, now in clinical trials. However, few activities to date have focused on the study of antisense per se. Further, few conceptual perspectives have regarded antisense as an integral part of cellular function and genetic regulation. The Ringberg conference showcased a number of systems that would seem unrelated if we regard antisense as a superficial tool to be imposed on nature. On the other hand, if we want to begin to regard antisense as a field of its own with deeper biological and genetic rationales, the Ringberg meeting provided much tantalizing evidence to do so.

Folate responsiveness during growth and development of Dictyostelium: separate but related pathways control chemotaxis and gene regulation.

Folate-controlled gene expression and chemotaxis have been examined in Dictyostelium wild-type and mutant strains. We show that regulation of the discoidin genes is sensitive to folate in growing cells as well as in suspension development. The signal is transferred via the N10-methylfolate-sensitive folate receptor sites, which also appear to confer the chemotactic response. The strain HG5145 has previously been isolated as a mutant that does not display chemotactic movement towards folate. Nevertheless, these cells are fully functional in folate-mediated downregulation of discoidin I expression. The strain ga 93 has been isolated as an overproducer mutant of the cyclic nucleotide phosphodiesterase inhibitor. Simultaneously, these cells fail to downregulate discoidin I in response to folate but are fully functional in folate chemotaxis. Therefore we conclude that the pathways for chemotaxis and for gene regulation diverge downstream of a common receptor type.

What makes an mRNA anti-sense-itive?

Antisense RNA has been used for some time as a versatile tool for silencing gene expression. There is ample evidence for gene regulation by endogenous antisense transcripts in prokaryotes and increasing insight into the molecular mechanisms underlying such regulation. The introduction of antisense gene constructs into eukaryotes has now become routine but the mechanisms by which gene expression is inhibited are barely understood. In recent years, several examples of endogenous eukaryotic antisense transcripts have been discovered, some of which probably serve regulatory functions. Here we will discuss a model to explain mechanisms of antisense-mediated gene silencing.