Drosophila Mitotic Genes Figure 1. The functions of 155 Drosophila genes detected by an RNAi screen of 1000 genes enriched in mitotic functions by co- expression analysis. (A) Distribution of 164 known mitotic genes in co-expression lists with cid, glu, eb1, zw10, ida and sti (Citron kinase); ‘consensus’, is a consensus co-expression list constructed by combining the six single gene lists. Numbers in columns are percentages. (B) Quantitative grouping of the 155 genes detected in the screen according to the observed RNAi phenotypes. (C) Distribution of the 155 genes in the consensus co-expression list; note that the frequency of mitotic genes decreases with the increase of the rank in the list. doi:10.1371/journal.pgen.1000126.g001 Tables S5 and S6). As shown in Figure 4, we found 44 dsRNAs that significantly increase the frequency of spontaneous chromo- some aberrations. Of the 44 genes identified by these dsRNAs, 38 have apparent human orthologs (Figure 4C) yet only 4 have previously been implicated in the maintenance of chromosome integrity (Table S6). These genes can be subdivided in several broad classes, based on their putative functions: (1) genes required for DNA replication, including Ribonucleotide reductase (RnrS), DNA primase (DNAprim), DNA polymerase alpha (DNApol), Orc5, RfC40, Rpa70/RPA1 and peterpan (ppan); (2) genes involved in both DNA replication and repair, such as mus209/PCNA, cul-4, thymidilate synthetase (Ts) and CG6854/CTP synthase; (3) genes that mediate different aspects of DNA repair but are not known to participate in DNA replication, such as DDB1, okra/RAD54L, CG6197/XAB2 and CG7003/MSH6; (4) genes involved in transcription and RNA maturation, including Dp/TFPD2, CG10354/XRN2, Taf6, l(2)NC136/CNOT3, noi/SF3A3, CG7757/PRPF3), without children (woc), CG6480/FRG1 and CG6686/SART1 (Table S6). Chromo- some aberrations were also induced by RNAi against 8 genes whose diverse functions are not easily classified into the four groupings above. These include BEAF-32, that encodes a chromatin insulator factor; dnk, that specifies a deoxyribonucleo- tide kinase similar to the human mitochondrial kinase TK2; Su(var)2-10, whose product is an E3 SUMO ligase; the H3.3B histone variant gene; SMC1, that encodes a conserved cohesin involved in the Cornelia de Lange syndrome in humans; Dcp-1, that specifies a caspase precursor; Megator (Mtor) that encodes a component of the putative spindle matrix; and CG17446, whose product is homologous to a subunit of the mammalian Set1 histone methyltransferase complex (Table S6). Our screen also identified 12 chromosome stability genes without any assigned putative functions, 7 of which are conserved in humans. Together, these results indicate that the maintenance of chromosome stability requires a large number of functions, many of which remain to be identified. The analysis of tubulin-stained mitotic figures revealed an interesting phenotype associated with the presence of chromosome aberrations. We observed many metaphase figures with the centric portions of broken chromosomes aligned at the metaphase plate and the acentric fragments near the cell poles (Figure 4B). Immuno- staining for the kinetochore marker Cenp-C [27] verified that most chromosome fragments at the poles of these metaphases were indeed devoid of centromere (Figure 4B). This phenotype suggests that chromosome fragments severed from their kinetochores are transported to the cell poles. A similar phenomenon has been observed in plants (Hemanthus) and in crane fly spermatocytes. In both systems, when a metacentric chromosome is cut with the laser, the resultant acentric fragment moves to the closest cell pole at the same velocity as anaphase chromosomes [28,29]. To explain this phenomenon, it has been suggested that the acentric chromosomes fragments adhere to the lateral surfaces or plus ends of microtubules and are transported poleward by the microtubule flux [29]. We believe that this mechanism also occurs in Drosophila S2 cells. Strong support for this view comes from observations on RPA70-depleted cells, which exhibit extreme chromosome fragmentation but form regular spindles. In these cells, most acentric fragments accumulate at the poles of ana/telophase figures, suggesting that they are driven poleward by microtubule-based forces (Figure 4B). Genes Required for Accurate Chromosome Segregation We identified 41 genes required for regular chromosome segregation. These genes are not required for spindle formation, PLoS Genetics | www.plosgenetics.org 3 July 2008 | Volume 4 | Issue 7 | e1000126