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.

An integrated assessment of resource use strategies in the Borana pastoral system of southern Ethiopia

In the pastoral production systems, mobility remains the main technique used to meet livestock’s fodder requirements. Currently, with growing challenges on the pastoral production systems, there is urgent need for an in-depth understanding of how pastoralists continue to manage their grazing resources and how they determine their mobility strategies. This study examined the Borana pastoralists’ regulation of access to grazing resources, mobility practices and cattle reproductive performances in three pastoral zones of Borana region of southern Ethiopia. The central objective of the study was to contribute to the understanding of pastoral land use strategies at a scale relevant to their management. The study applied a multi-scalar methodological approach that allowed zooming in from communal to individual herd level. Through participatory mapping that applied Google Earth image print out as visual aid, the study revealed that the Borana pastoralists conceptualized their grazing areas as distinctive grazing units with names, borders, and specific characteristics. This knowledge enables the herders to communicate the condition of grazing resources among themselves in a precise way which is important in management of livestock mobility. Analysis of grazing area use from the participatory maps showed that the Borana pastoralists apportion their grazing areas into categories that are accessed at different times of the year (temporal use areas). This re-organization is an attempt by the community to cope with the prevailing constraints which results in fodder shortages especially during the dry periods. The re-organization represents a shift in resource use system, as the previous mobility practice across the ecologically varied zones of the rangelands became severely restricted. Grazing itineraries of 91 cattle herds for over 16 months obtained using the seasonal calendar interviews indicated that in the areas with the severest mobility constraints, the herders spent most of their time in the year round use areas that are within close proximity to the settlements. A significant change in mobility strategy was the disallowing of foora practice by the communities in Dirre and Malbe zones in order to reduce competition. With the reduction in mobility practices, there is a general decline in cattle reproductive parameters with the areas experiencing the severest constraints showing the least favourable reproductive performances. The study concludes that the multi-scalar methodology was well suited to zoom into pastoral grazing management practices from communal to individual herd levels. Also the loss of mobility in the Borana pastoral system affects fulfilment of livestock feed requirements thus resulting in reduced reproductive performances and herd growth potentials. While reversal of the conditions of the situations in the Borana rangelands is practically unfeasible, the findings from this research underscore the need to protect the remaining pastoral lands since the pastoral production system remains the most important livelihood option for the majority of the Borana people. In this regards the study emphasises the need to adopt and domesticate regional and international policy frameworks such as that proposed by the African Union in 2010.