Drosophila Mitotic Genes Figure 8. Mitotic phenotypes observed in the CS4 and CS5 phenoclusters. In merges, DNA is blue and tubulin green. (A) In RNAi cells of the CS4 phenocluster, sister chromatids do not separate so that the chromosomes remain at the center of the cell, while the spindles elongate and assume morphologies typical of ana/telophase figures. Top panels, anaphase-like and telophase-like spindles from Cyclin B RNAi cells. Bottom panels, anaphase-like and telophase-like spindles from U2A9 (splicing factor) RNAi cells. Note that chromosome arms are parallel to the spindle axis (see text for explanation). (B) Mitotic figures from the CS5 phenocluster. In both CG3221 and CG5649 RNAi cells, the two sets of segregating chromosomes remain close to each other and fail to reach the spindle poles (compare with control cells in Figure 4A). Meta, metaphase; ana, anaphase; telo, telophase. Scale bars, 5 mm. doi:10.1371/journal.pgen.1000126.g008 Genes Required to Maintain Proper Chromosome Structure We identified 11 dsRNAs that cause defects in chromosome structure without affecting spindle assembly. The phenotypes produced by these RNAs can be grouped into three phenoclusters we call CC1, CC2 and CC3 (Figure 2). The CC1 group includes Minichromosome maintenance 3 (Mcm3), Mcm7 and cap. Mcm3 and Mcm7 encode the orthologs of two components of the human (MCM)2-7 helicase complex (Table S6), while Cap encodes a protein orthologous to the SMC3 cohesin whose mutant form is responsible for a mild variant of the Cornelia de Lange syndrome [43]. RNAi for these genes resulted in loss of sister chromatid cohesion in the heterochromatic regions of the chromosomes and defective chromosome congression and segregation (Figure 9A and Figure S9). A similar phenotype was previously observed in mutants in the wings-apart like (wapl) Drosophila gene [44]; the human ortholog of Wapl interacts with cohesin and regulates its association with chromatin [45,46]. The CC2 phenocluster includes 5 genes that encode well-known condensins: SMC2, Gluon/SMC4, CapD2, Cap-G and Barren/ CAP-H (Figure 2 and Table S6). RNAi for these genes resulted in very similar phenotypes. In all cases, chromosomes displayed an abnormal mitotic condensation: although their longitudinal axis was shortened normally, their sister chromatids were swollen and fuzzy. In addition, ana/telophase figures displayed frequent lagging chromosomes and chromatin bridges, consistent with a strong defect in sister chromatid resolution during anaphase (Figure S10). In contrast, in RNAi cells for either gene in the CC3 group, Topoisomerase II (Top2), greatwall (gwl) (encoding a conserved kinase; see Table S6) and Orc-2, metaphase chromosomes were abnor- PLoS Genetics | www.plosgenetics.org 10 July 2008 | Volume 4 | Issue 7 | e1000126