Human centromere reconstitution in a heterologous system

The centromere is the region on a chromosome by which a chromosome is attached to spindle fibres and is responsible for the division of chromosomes during mitosis. Centromere location is not determined by DNA sequence alone, but in most eukaryotes centromere identity is specified by the presence of a centromere-specific histone H3 variant, CENP-A. The Heun lab has established a lacO/lacI tethering system to target LacI fused to human CENP-A to lacO inserted at regions throughout the drosophila genome. Through utilizing Golden Gate cloning to generate vectors for co-expression of the components of the remaining human centromere components and insertion of lacO tethering sites at specified points in the drosophila genome I hope to reconstitute a functional human centromere. By utilizing a heterologous system where there is no cross-reactivity of the centromere forming components between drosophila and humans it is possible to determine the minimal components necessary for centromere formation.