Selfish DNA: a pharmaceutical perspective.

Almost 25 years ago, Theo Dingermann published the discovery of a new mobile genetic element in the unicellular microbe Dictyostelium discoideum in the journal Science. An interesting property of this new molecular parasite, the Dictyostelium Repetitive Element (DRE), was that all integrations were found approximately 50 base pairs (bp) upstream of transfer RNA (tRNA) genes in the D. discoideum genome, thus implying an active targeting mechanism to avoid the disruption of host cell genes by the retrotransposition process. Since then, the facultative multicellular "social amoeba" D. discoideum has become a popular model for analyzing complex cellular functions such as cell movement, chemotaxis, phagocytosis, and cell differentiation, important areas of biomedical research that are often hard to investigate in cells from "higher organisms" including humans. Therefore, progress in the development of methods to study Dictyostelium biology has also provoked research on transposable elements in this organism. Early work on the DRE element suggested that studying its molecular mechanism of site-specific integration might promote human gene therapy technology through the design of integrating gene transfer vectors with low intrinsic genotoxic potential. In this review article, I will briefly review the original research performed on the DRE transposable element in the Dingermann lab and report on how the emergence of genomics technologies and the development of tools to analyze de novo retrotransposition events in D. discoideum cells will expand our knowledge of DRE biology in the future.

Transfer RNA gene-targeted integration: an adaptation of retrotransposable elements to survive in the compact Dictyostelium discoideum genome.

Almost every organism carries along a multitude of molecular parasites known as transposable elements (TEs). TEs influence their host genomes in many ways by expanding genome size and complexity, rearranging genomic DNA, mutagenizing host genes, and altering transcription levels of nearby genes. The eukaryotic microorganism Dictyostelium discoideum is attractive for the study of fundamental biological phenomena such as intercellular communication, formation of multicellularity, cell differentiation, and morphogenesis. D. discoideum has a highly compacted, haploid genome with less than 1 kb of genomic DNA separating coding regions. Nevertheless, the D. discoideum genome is loaded with 10% of TEs that managed to settle and survive in this inhospitable environment. In depth analysis of D. discoideum genome project data has provided intriguing insights into the evolutionary challenges that mobile elements face when they invade compact genomes. Two different mechanisms are used by D. discoideum TEs to avoid disruption of host genes upon retrotransposition. Several TEs have invented the specific targeting of tRNA gene-flanking regions as a means to avoid integration into coding regions. These elements have been dispersed on all chromosomes, closely following the distribution of tRNA genes. By contrast, TEs that lack bona fide integration specificities show a strong bias to nested integration, thus forming large TE clusters at certain chromosomal loci that are hardly resolved by bioinformatics approaches. We summarize our current view of D. discoideum TEs and present new data from the analysis of the complete sequences of D. discoideum chromosomes 1 and 2, which comprise more than one third of the total genome.

Template jumping by a LINE reverse transcriptase has created a SINE-like 5S rRNA retropseudogene in Dictyostelium.

Short interspersed nuclear elements (SINEs) are non-autonomous retroelements that mimic the 3' ends of so-called long interspersed nuclear elements (LINEs) to ensure their propagation by proteins encoded by autonomous LINEs. The Dictyostelium discoideum genome contains a family of LINE-like retrotransposons that specifically target tRNA genes for integration (TRE elements). We describe here a retrotransposed ribosomal 5S RNA pseudogene in the D. discoideum genome that contains at its 3' end an 8-bp sequence derived from the 3' end of a TRE and a polyadenine tail. The r5S "retropseudogene" is flanked by target-site duplications that are characteristic for TREs, and is inserted upstream of a tRNA gene, just like a typical TRE. The D. discoideum r5S retropseudogene has structural features of a SINE, but has not been amplified, probably due to the 5'-truncation that occurred upon its initial retrotransposition. The discovery of this D. discoideum r5S retropseudogene reveals that SINEs can be created de novo during reverse transcription of LINE transcripts, if the LINE-encoded reverse transcriptase dissociates from the LINE RNA and jumps to other cellular RNAs-particularly genes transcribed by RNA polymerase III-to create continuous mixed cDNAs.

Effects of medicinal compounds on the differentiation of the eukaryotic microorganism dictyostelium discoideum: can this model be used as a screening test for reproductive toxicity in humans?

Dictyostelium discoideum is a single-cell, eukaryotic microorganism that can undergo multicellular development in order to produce dormant spores. We investigated the capacity of D. discoideum to be used as a rapid screening system for potential developmental toxicity of compounds under development as pharmaceuticals. We used a set of four transgenic D. discoideum strains that expressed a reporter gene under the control of promoters that are active at certain time periods and in distinct cell types during D. discoideum development. We found that teratogens such as valproic acid, tretinoin, or thalidomide interfered to various extents with D. discoideum development, and had different effects on prestalk and prespore cell-specific reporter gene expression. Phenytoin was inactive in this assay, which may point to limitations in metabolization of the compound in Dictyostelium required to exert developmental toxicity. D. discoideum cell culture is cheap and easy to handle compared to mammalian cell cultures or animal teratogenicity models. Although the Dictyostelium-based assay described in this report may not securely predict the teratogenic potential of these drugs in humans, this organism may be qualified for rapid large-scale screenings of synthetic compounds under development as new pharmaceuticals for their potential to interfere with developmental processes and thus help to reduce the amount of teratogenicity tests in animal models.

Dictyostelium mobile elements: strategies to amplify in a compact genome.

Dictyostelium discoideum is a eukaryotic microorganism that is attractive for the study of fundamental biological phenomena such as cell-cell communication, formation of multicellularity, cell differentiation and morphogenesis. Large-scale sequencing of the D. discoideum genome has provided new insights into evolutionary strategies evolved by transposable elements (TEs) to settle in compact microbial genomes and to maintain active populations over evolutionary time. The high gene density (about 1 gene/2.6 kb) of the D. discoideum genome leaves limited space for selfish molecular invaders to move and amplify without causing deleterious mutations that eradicate their host. Targeting of transfer RNA (tRNA) gene loci appears to be a generally successful strategy for TEs residing in compact genomes to insert away from coding regions. In D. discoideum, tRNA gene-targeted retrotransposition has evolved independently at least three times by both non-long terminal repeat (LTR) retrotransposons and retrovirus-like LTR retrotransposons. Unlike the nonspecifically inserting D. discoideum TEs, which have a strong tendency to insert into preexisting TE copies and form large and complex clusters near the ends of chromosomes, the tRNA gene-targeted retrotransposons have managed to occupy 75% of the tRNA gene loci spread on chromosome 2 and represent 80% of the TEs recognized on the assembled central 6.5-Mb part of chromosome 2. In this review we update the available information about D. discoideum TEs which emerges both from previous work and current large-scale genome sequencing, with special emphasis on the fact that tRNA genes are principal determinants of retrotransposon insertions into the D. discoideum genome.

On the problem of establishing the subcellular localization of Dictyostelium retrotransposon TRE5-A proteins by biochemical analysis of nuclear extracts.

At first sight a protein that is enriched in extracts prepared from nuclei by means of biochemical methods can be considered to be a nuclear protein in vivo. Although this assumption will hold true for most of the analyzed proteins, it could also lead to false interpretations. We analyzed the subcellular distribution of endogenous and plasmid-borne proteins derived from the retrotransposon TRE5-A of Dictyostelium discoideum. In biochemical fractionation experiments the proteins encoded by TRE5-A open reading frame 1 (ORF1p) and the putative endonuclease encoded in ORF2 (ENp) were found in the detergent-insoluble material containing the nuclei. However, salt extraction of isolated nuclei did not considerably release the TRE5-A proteins. Instead, the TRE5-A proteins were strongly enriched in a fraction that contained the chromosomal DNA after removal of most cytoskeletal and histone proteins. These observations implied that ORF1p and ENp were both attached to chromatin in vivo, but this conclusion was disproved by the expression of genetic fusions of green fluorescent protein with either ORF1p or ENp. We show conclusive evidence that both fusion proteins were located as large aggregates of native protein in the cytoplasm of D. discoideum cells.

The complex repeats of Dictyostelium discoideum.

In the course of determining the sequence of the Dictyostelium discoideum genome we have characterized in detail the quantity and nature of interspersed repetitive elements present in this species. Several of the most abundant small complex repeats and transposons (DIRS-1; TRE3-A,B; TRE5-A; skipper; Tdd-4; H3R) have been described previously. In our analysis we have identified additional elements. Thus, we can now present a complete list of complex repetitive elements in D. discoideum. All elements add up to 10% of the genome. Some of the newly described elements belong to established classes (TRE3-C, D; TRE5-B,C; DGLT-A,P; Tdd-5). However, we have also defined two new classes of DNA transposable elements (DDT and thug) that have not been described thus far. Based on the nucleotide amount, we calculated the least copy number in each family. These vary between <10 up to >200 copies. Unique sequences adjacent to the element ends and truncation points in elements gave a measure for the fragmentation of the elements. Furthermore, we describe the diversity of single elements with regard to polymorphisms and conserved structures. All elements show insertion preference into loci in which other elements of the same family reside. The analysis of the complex repeats is a valuable data resource for the ongoing assembly of whole D. discoideum chromosomes.

Gene function analysis by amber stop codon suppression: CMBF is a nuclear protein that supports growth and development of Dictyostelium amoebae.

The C-module-binding factor, CMBF, is a nuclear DNA-binding protein which was originally identified through its specific binding to a promoter element within the retrotransposable element TRE5-A of Dictyostelium discoideum AX2 cells. In order to analyse putative physiological functions of CMBF for the TRE5-A-hosting D. discoideum cells, we used a novel strategy to create mutant cell lines which stably underexpressed functional CMBF. An amber (UAG) translation stop codon was introduced into the chromosomal copy of the CMBF-encoding gene (cbfA), and an amber suppressor tRNA gene was expressed in the same mutant cells. Due to the low efficiency of translation stop codon suppression in this system all recovered cell lines expressed <20 % of wild-type CMBF levels. The mutant cell lines displayed strong growth phenotypes when plated on their natural food source, bacteria. We show evidence that growth reduction was due to impaired phagocytosis of bacteria in the mutants. All obtained mutants showed a strong developmental defect which was defined by the formation of very small fruiting bodies. The strength of the developmental phenotype appeared to depend upon the residual CMBF levels maintained in the mutants. We propose that CMBF is a general transcription regulator which supports the normal expression of several genes required for the maintenance of high proliferation rates of D. discoideum amoebae as well as proper aggregation and development. Our results demonstrate that amber stop codon suppression may be a useful strategy to stably underexpress proteins whose coding genes cannot be successfully disrupted by homologous recombination.

A Dictyostelium protein binds to distinct oligo(dA) x oligo(dT) DNA sequences in the C-module of the retrotransposable element DRE.

The genome of the eukaryotic microbe Dictyostelium discoideum contains some 200 copies of the nonlong-terminal repeat retrotransposon DRE. Among several unique features of this retroelement, DRE is transcribed in both directions leading to the formation of partially overlapping plus strand and minus strand RNAs. The synthesis of minus strand RNAs is controlled by the C-module, a 134-bp DNA sequence located at the 3'-end of DRE. A nuclear protein (CMBF) binds to the C-module via interaction with two almost homopolymeric 24 bp oligo(dA) x oligo(dT) sequences. The DNA-binding drugs distamycin and netropsin, which bind to A x T-rich DNA sequences in the minor groove, competed efficiently for the binding of CMBF to the C-module. The CMBF-encoding gene, cbfA, was isolated and a DNA-binding domain was mapped to a 25-kDa C-terminal region of the protein. A peptide motif involved in the binding of A x T-rich DNA by high mobility group-I proteins ('GRP' box) was identified in the deduced CMBF protein sequence, and exchange of a consensus arginine residue for alanine within the CMBF GRP box abolished the interaction of CMBF with the C-module. The current data support the theory that CMBF binds to the C-module by detecting its long-range DNA conformation and interacting with A x T base pairs in the minor groove of oligo(dA) x oligo(dT) stretches.

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

Retrotransposable elements are genetic enti ties which move and replicate within host cell genomes We have previously reported on the structures and ge nomic distributions of two non-long terminal repea (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.

Identification of Melissa officinalis Subspecies by DNA Fingerprinting.

The random amplified polymorphic DNA analysis (RAPD) is a method to study genetic variability within and between populations and species on the basis of the amplification of anonymous fragments from genomic DNA templates by means of polymerase chain reaction (PCR). We applied RAPD analysis in order to distinguish medicinal plant subspecies at the level of their genomes. In this study we investigated various samples of two MELISSA subspecies and showed that RAPD analysis is a fast and reliable method to distinguish subspecies on the pharmaceutical market that have been previously classified according to the distribution pattern of compounds present in the lemon balm oil.

Characterization of Echinacea species and detection of possible adulterations by RAPD analysis.

In this report we describe the discrimination of all three pharmaceutically relevant Echinacea species by random amplified polymorphic DNA (RAPD) analysis. Genomic DNA prepared from either fresh root material or dried roots was probed in polymerase chain reactions (PCR) using several arbitrary primers. Distinct banding patterns were obtained for E. purpurea, E. angustifolia var. angustifolia, and E. pallida, respectively. Individual Echinacea drugs could be identified within mixtures containing variable amounts of individual drugs. We propose to use RAPD analysis as a rapid and reliable test for proving the identity of Echinacea root drugs.

Retrotransposable elements in the Dictyostelium discoideum genome.

Repetitive DNA is a major component of any living cell. In eukaryotes retrotransposable elements make up several percent of the genome size, and consequently, retroelements are often identified in experiments aimed at establishing physical maps and whole genome sequences. In this review, recent progress in the characterization of retrotransposable elements in the genome of the eukaryotic microorganism Dictyostelium discoideum is summarized with a focus on retroelements which integrate near transfer RNA genes with intriguing position specificity.

Tdd-3, a tRNA gene-associated poly(A) retrotransposon from Dictyostelium discoideum.

The full-length 5218-bp sequence of the mobile genetic element Tdd-3 from Dictyostelium discoideum is described. Tdd-3 encodes two overlapping open reading frames (ORFs) flanked by non-redundant, untranslated regions. The deduced amino acid sequence of ORF2 is homologous to reverse transcriptases (RTs) encoded by the class of poly(A) retrotransposons. ORF2 also encodes a putative protein domain related to the family of apurinic/apyrimidinic (AP) endonucleases, whose retroelement-encoded homologs have recently been proposed to represent the integrase function of poly(A) retrotransposons. Comparison of several genomic Tdd-3 copies revealed that element insertion is orientation specific and occurs about 100 bp downstream of tRNA genes in the D. discoideum genome. These properties of Tdd-3 suggest that the element is a tRNA gene-associated poly(A) retroelement present in the D. discoideum genome. Analysis of several cloned cDNAs derived from Tdd-3-specific plus strand RNAs indicate that the element is transcribed and polyadenylated during the growth of D. discoideum cells.

Evaluation of the Teratogenic Potential of Valproic Acid Analogues in Transgenic Dictyostelium discoideum Strains.

Very early during the development of new pharmaceuticals toxicological tests are most important. In addition to acute and chronic toxicity tests, it is crucial to estimate the teratogenic potential of promising drugs. We established a simple biological test system based on the cellular slime mold Dictyostelium discoideum. Under certain environmental conditions single cells of D. discoideum aggregate and undergo a relatively simple cell differentiation program, leading to the formation of stalk and spore cells. Transgenic D. discoideum strains carrying the bacterial beta-galactosidase gene under the control of various developmentally regulated D. discoideum promoters were shown to be useful tools to test the teratogenic potential of valproic acid (VPA). This study describes the effects of the VPA analogues S-4-yn-VPA, R-4-yn-VPA, and 2-ethyl-4-pentynoic acid on the D. discoideum developmental system. The presence of S-4-yn-VPA during D. discoideum development resulted in a strong inhibition of spore cell differentiation, whereas stalk cell formation was less affected. The enantiomer R-4-yn-VPA as well as 2-ethyl-4-pentynoic acid had only moderate effects on D. discoideum development. The above results are consistent with data obtained in mammalian teratogenicity assays, and suggest that D. discoideum development should be investigated with a number of additional substances to provide a simple alternative for high throughput screenings of new drugs.

Molecular cloning of a cDNA encoding the nucleosome core histone H3 from Dictyostelium discoideum by genetic screening in yeast.

The one-hybrid method for genetic screening in yeast was used to search a Dictyostelium discoideum cDNA library for DNA-binding proteins that interact with the C-module of the Dictyostelium repetitive element. The C-module was formerly shown to contain two high affinity, sequence-specific binding sites for a nuclear protein factor of unknown function (CMBF). The bait DNA sequence was bound in vivo by a cDNA-encoded protein whose derived amino acid sequence showed high homology to nucleosome core histone H3, but not to partially available CMBF sequences. The D. discoideum histone H3 homolog is encoded by a single gene and shows significant sequence variation at the amino terminus of the protein, including a triple-serine insertion not found in any other histone H3.

Developmentally regulated promoters from Dictyostelium discoideum as molecular markers for testing potential teratogens.

Already very early in the course of the development of new pharmaceutically relevant drugs toxicological tests are most important. In addition to acute and chronic toxicity the estimation of the teratogenic potential is rather crucial. We have recently shown that the eukaryotic microorganism Dictyostellium discoideum is a useful organism to test the cytotoxicity of chemical compounds. Since D. discoideum is competent of undergoing both vegetative growth and development, further investigations were aimed to establish a D. discoideum-based test system which could predict possible interference of drugs with developmental programs. We developed a method which allows to detect and to quantify effects of possible teratogens on D. discoideum development. This method is based on different transgenic D. discoideum strains, each carrying a bacterial lacZ gene under the control of a distinct developmentally regulated D. discoideum promoter. Here we describe the effects of the known teratogenic compound valproic acid (VPA) on this system.

A nuclear protein factor binds specifically to the 3'-regulatory module of the long-interspersed-nuclear-element-like Dictyostelium repetitive element.

The Dictyostelium repetitive element DRE integrates in a position-specific manner upstream of tRNA genes in the Dictyostelium discoideum genome. DRE has structural similarities to the group of long interspersed nuclear elements, whose replication mechanism is poorly understood. The C-module at the 3' end of DRE encodes a regulatory cis-acting sequence that contains an RNA polymerase II promoter. This promoter directs the synthesis of RNAs that are thought to play a critical role in DRE transposition. In this study, we describe the identification of a nuclear protein factor that binds to the C-module in a sequence-specific manner. The C-module-binding factor (CMBF) recognizes three DNA sequence motifs that contain homopolymeric (dT) stretches of variable lengths, but does not bind to a standard RNA polymerase II promoter from D, discoideum. Analysis of highly CMBF-enriched fractions and glycerol gradient sedimentation of CMBF suggest that the factor exists as a monomeric 115-kDa protein. Possible roles of CMBF in DRE transposition are discussed.

A simple cytotoxicity assay using the eucaryotic microorganism Dictyostelium discoideum.

First attempts to evaluate the potential of the cellular slime mold Dictyostelium discoideum as a model system for cytotoxic tests of potential drugs are described. Using the cell counter and analyser system, CASY 1, two parameters, cell count and mean cell volume, could be established as relevant parameters to estimate cytotoxicity. The effects on these two parameters of two well characterized drugs, the gentamycin analogue G418 and doxorubicin, are reported.