Nuclear organisation and epigenetic regulation of gene expression in Dictyostelium discoideum

A series of vectors for the over-expression of tagged proteins in Dictyostelium were designed, constructed and tested. These vectors allow the addition of an N- or C-terminal tag (GFP, RFP, 3xFLAG, 3xHA, 6xMYC and TAP) with an optimized polylinker sequence and no additional amino acid residues at the N or C terminus. Different selectable markers (Blasticidin and gentamicin) are available as well as an extra chromosomal version; these allow copy number and thus expression level to be controlled, as well as allowing for more options with regard to complementation, co- and super-transformation. Finally, the vectors share standardized cloning sites, allowing a gene of interest to be easily transfered between the different versions of the vectors as experimental requirements evolve. The organisation and dynamics of the Dictyostelium nucleus during the cell cycle was investigated. The centromeric histone H3 (CenH3) variant serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. A number of Dictyostelium histone H3-domain containing proteins as GFP-tagged fusions were expressed and it was found that one of them functions as CenH3 in this species. Like CenH3 from some other species, Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins. The targeting domain, comprising α-helix 2 and loop 1 of the histone fold is required for targeting CenH3 to centromeres. Compared to the targeting domain of other known and putative CenH3 species, Dictyostelium CenH3 has a shorter loop 1 region. The localisation of a variety of histone modifications and histone modifying enzymes was examined. Using fluorescence in situ hybridisation (FISH) and CenH3 chromatin-immunoprecipitation (ChIP) it was shown that the six telocentric centromeres contain all of the DIRS-1 and most of the DDT-A and skipper transposons. During interphase the centromeres remain attached to the centrosome resulting in a single CenH3 cluster which also contains the putative histone H3K9 methyltransferase SuvA, H3K9me3 and HP1 (heterochromatin protein 1). Except for the centromere cluster and a number of small foci at the nuclear periphery opposite the centromeres, the rest of the nucleus is largely devoid of transposons and heterochromatin associated histone modifications. At least some of the small foci correspond to the distal telomeres, suggesting that the chromosomes are organised in a Rabl-like manner. It was found that in contrast to metazoans, loading of CenH3 onto Dictyostelium centromeres occurs in late G2 phase. Transformation of Dictyostelium with vectors carrying the G418 resistance cassette typically results in the vector integrating into the genome in one or a few tandem arrays of approximately a hundred copies. In contrast, plasmids containing a Blasticidin resistance cassette integrate as single or a few copies. The behaviour of transgenes in the nucleus was examined by FISH, and it was found that low copy transgenes show apparently random distribution within the nucleus, while transgenes with more than approximately 10 copies cluster at or immediately adjacent to the centromeres in interphase cells regardless of the actual integration site along the chromosome. During mitosis the transgenes show centromere-like behaviour, and ChIP experiments show that transgenes contain the heterochromatin marker H3K9me2 and the centromeric histone variant H3v1. This clustering, and centromere-like behaviour was not observed on extrachromosomal transgenes, nor on a line where the transgene had integrated into the extrachromosomal rDNA palindrome. This suggests that it is the repetitive nature of the transgenes that causes the centromere-like behaviour. A Dictyostelium homolog of DET1, a protein largely restricted to multicellular eukaryotes where it has a role in developmental regulation was identified. As in other species Dictyostelium DET1 is nuclear localised. In ChIP experiments DET1 was found to bind the promoters of a number of developmentally regulated loci. In contrast to other species where it is an essential protein, loss of DET1 is not lethal in Dictyostelium, although viability is greatly reduced. Loss of DET1 results in delayed and abnormal development with enlarged aggregation territories. Mutant slugs displayed apparent cell type patterning with a bias towards pre-stalk cell types.

Analyzing Gene Centres with the Help of the Checklist Method – the Case of Syria

The present survey of species diversity of cultivated plants is the first for Syria. Some cultivated species will be added in the future, because due to the civil war in Syria, it was not possible to visit the country in the frame of the present work, as initially planned. Checklists proved to be a useful tool for overviewing the cultivated plants of selected areas and allow a characterization of the state of plant genetic resources of Syria. Syria has experienced several civilizations. Man settled in this productive land since ancient times and used its resources. However, such use has led to changes in vegetation and decline of wildlife through the country, in seashore areas, interior, mountains, and grassland. Plant domestication and growing started more than 10,000 years ago in West Asia. Since then, plentiful of economic plant species were present and used by man and his domesticated animals. Forming a part of the Fertile Crescent, where many of the world’s agricultural plants have evolved, Syria is extremely rich in agrobiodiversity. Wild progenitors of wheat and barley and wild relatives of many fruit trees such as almonds and pistachio as well as forage species are still found in marginal lands and less disturbed areas. These are threatened by a wide range of human activities, notably modern, extensive agriculture, overgrazing, overcutting and urban expansion. Syria is also considered as part of one of the main centres of origin, according to Vavilov, who had collected in Syria in 1926. The first expeditions to crop fields showed the exclusive nature of cultivated plants in Syria with a high number of endemic forms. Furthermore, Syria is a part of a biodiversity hotspot. Several studies have been performed to study agrobiodiversity in different parts of Syria, but usually on wild species. Many collections have been carried out; however, they focussed preferably on cereals and pulses, and particularly on wheat, like Vavilov’s expedition. Only 30 crops make up the major part of the conserved Syrian crop plant material in the genebank, indicating that most of the remaining 7,000 species of cultivated plants and many other valuable genetic resources species have only been included on a limited scale in the genebank collections. Although a small country (185,180 km2), Syria accommodates numerous ecosystems that allow for a large diversity of plant genetic resources for agriculture ranging from cold-requiring to subtropical crops to live and thrive. Only few references are available in this respect. The aim of the present study was to complete a checklist of Syria’s cultivated plants of agriculture and horticulture excluding plants only grown as ornamental or for forestry. Furthermore, plants taken for reforestation have not been included, if they do not have also agricultural or horticultural uses. Therefore, the inclusion of plants into the checklist follows the same principles as “Mansfeld’s Encyclopedia”. Main sources of information were published literature, floras of Syria, Lebanon and the Mediterranean, as well as Syrian printed sources in Arabic and/or English, reports from FAO on agricultural statistics in Syria, and data from ICARDA and Bioversity International. In addition, personal observations gathered during professional work in the General Commission for Scientific Agricultural Research (GCSAR) in Syria (since 1989) and participation in projects were taken into account. These were: (1) A project on “Conservation and Sustainable Use of Dry Land Agrobiodiversity in the Near East” with participation of Jordan, Lebanon, Syria, and the Palestinian Authority, focussing on landraces and wild relatives of barley, wheat, lentil, alliums, feed legumes, and fruit trees (1999–2005). (2) A project for vegetable landraces (1993–1995) in collaboration with the former International Plant Genetic Resources Institute and the UN Development Programme, in which 380 local vegetable accessions were evaluated. For medicinal plants and fruit trees I was in personal contact with departments of GCSAR and the Ministry of Agriculture and Agrarian Reform, as well as with private organizations. The resulting checklist was compared with the catalogues of crop plants of Italy and a checklist of cultivated plants of Iraq. The cultivated plant species are presented in alphabetical order according to their accepted scientific names. Each entry consists of a nomenclatural part, folk names, details of plant uses, the distribution in Syria (by provinces), a textual description, and references to literature. In total, 262 species belonging to 146 genera and 57 families were identified. Within-species (intraspecific) diversity is a significant measure of the biodiversity. Intraspecific diversity for wild plants has been and remains to be well studied, but for crop plants there are only few results. Mansfeld’s method is an actual logical contribution to such studies. Among the families, the following have the highest number of crop species: Leguminosae (34 spp.), Rosaceae (24), Gramineae (18), Labiatae (18), Compositae (14), Cruciferae (14), Cucurbitaceae (11), Rutaceae (10), Malvaceae (9), Alliaceae (7), and Anacardiaceae (7). The establishment of an effective programme for the maintenance of plant genetic resources in Syria started in the mid-1970s. This programme considered ex situ and in situ collection of the genetic resources of various field crops, fruit trees and vegetables. From a plant genetic resources viewpoint, it is clear that the homegarden is an important location for the cultivation of so-called neglected and underutilized species (neglected from a research side and underutilized from a larger economic side). Such species have so far not received much care from ecologists, botanists and agronomists, and they are considerably under-represented in genebanks.