Supplementary MaterialsFigure S1: Precocious sister chromatid separation and faulty chromosome segregation following RNAi for and RNAi cells. after RNAi for genes from the CS4 phenocluster. The chromosomes stay on the cell equator as the spindle elongates to suppose an ana/telophase-like morphology. The chromosomes at the guts from the cell decondense as occurs during normal telophase frequently. Level bar, 5 RNAi cells, chromosomes are extremely condensed and the sister chromatids are separated. However, sister chromatid separation (SCS) is not observed in RNAi cells with normally condensed chromosomes. We thus consider SCS as a secondary consequence of the excessive chromosome condensation, rather than a direct effect of gene product depletion. In support of this idea, RNAi for genes of Ganciclovir cost the CS2 phenocluster (Physique S1) causes sister chromatid separation regardless of the degree of chromosome condensation. Level SQLE bar, 5 m.(5.70 MB TIF) pgen.1000126.s007.tif (5.4M) GUID:?435C531D-ACDD-4060-8D99-A83B047C36CB Physique S8: Pole-to-pole spindle lengths (meanSE) of ana/telophase figures observed in the CS1-CS5 phenoclusters. The ana/telophase spindles observed in all the RNAi experiments included in the graph are significantly longer (p 0.001 in Student’s t-test) than control (ctr) spindles.(1.01 MB TIF) pgen.1000126.s008.tif (985K) Ganciclovir cost GUID:?A5C4980F-83D8-4B9B-A597-37A9977E2FCD Physique S9: RNAi for genes of the CC1 phenocluster results both in a lack of sister chromatid cohesion in the heterochromatic regions of the chromosomes and in defective chromosome segregation. Ctr, control; c-meta, colchicine/hypotonic-treated metaphase chromosomes. Level bar, 5 m.(3.47 MB TIF) pgen.1000126.s009.tif (3.3M) GUID:?75D16B1D-3D3B-4D6C-85A3-E7FEE32FB260 Figure S10: Defects in chromosome condensation and segregation observed after RNAi for genes of the CC2 phenocluster. Ctr, control; c-meta, colchicine/hypotonic-treated metaphase chromosomes. Note the considerable chromatin bridges in the ana/telophase figures. Level bar, 5 m.(3.37 MB TIF) pgen.1000126.s010.tif (3.2M) GUID:?57BBD2A4-4A3E-4A25-9DF2-6E141A64DC1A Physique S11: Monopolar spindles, short spindles and defective chromosome segregation observed after RNAi for and (SA1 phenocluster). The telophase-like figures of RNAi cells display abnormally long astral microtubules. Level bar, 5 (translation factor) and RNAi cells, the chromosomes remain at the center of the cells (as seen after RNAi for the CS4 group genes) and the centrosomes detach from your spindle poles. In RNAi cells, spindles are either monopolar or monastral bipolar. In cells with monastral bipolar spindles, chromosome segregation does not occur and the chromosomes remain associated with the astral poles. Level bar, 5 m.(3.93 MB TIF) pgen.1000126.s015.tif (3.7M) GUID:?25342801-30AE-4AAE-A194-A7CF958205C4 Physique S16: Abnormal chromosome condensation and multiple mitotic defects observed after RNAi for (SC1) and (SC2). Ctr, control; c-meta, colchicine/hypotonic-treated metaphase chromosomes. Note that in RNAi cells chromosomes are abnormally long and irregularly condensed. In contrast, Ganciclovir cost after RNAi for tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the capabilities of bioinformatics and RNAi technology to detect novel mitotic genes. We found that genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expressionCbased list of 1, 000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle business. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in Ganciclovir cost chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expressionCbased method for the detection of mitotic functions works amazingly well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins. Author Summary Mitosis is the evolutionarily conserved process that enables a dividing cell to equally partition its genetic material between the two child cells. The fidelity of mitotic division is crucial for normal development of multicellular organisms and to prevent malignancy or birth defects. Ganciclovir cost Understanding the molecular mechanisms of mitosis requires the identification of genes involved in this process. Previous studies.