This was necessary to be able to compare the conservation of novel hits versus known motifs

1/Ndc80 and appear to function normally, as determined by the ability of sister chromatids to migrate poleward at anaphase. This disagrees with previous reports that CPC activity is required for assembly of the outer kinetochore. Although the CPC does influence the bundling of midzone microtubules, a structurally distinct spindle midzone can form in the absence of the CPC, as shown by the movements of CENP-E. Thus, CENP-E must use a central spindle Digitoxin site targeting mechanism either distinct from or upstream from that of the CPC. It could be that successful cytokinesis requires subtle aspects of CPC localization that we are unable to detect in our present system. Alternatively, critical substrates, which may include vimentin, Mklp1, MgcRacGAP/Cyk4, and myosin II regulatory light chain, might simply require a higher level of kinase activity to be phosphorylated beyond a critical threshold. This could either be because of weaker binding to the kinase or because of more localized counteracting protein phosphatase activity. Transfer of the budding yeast CPC from centromeres to the overlapping microtubules of the central spindle requires CDC14 phosphatase. Interestingly, Cdc14 phosphatase is required for central spindle formation in Caenorhabditis elegans. The role of CDC14 isoforms in vertebrate cells is less clear and merits further study. 650 JCB VOLUME 187 NUMBER 5 2009 These experiments reveal a new complexity to CPC regulation in mitosis. Whether wild-type INCENP is able to activate aurora B to differing extents at different cellular locations in vivo remains to be determined. It is possible that interactions of INCENP with Cdk1 or Plk1 could contribute to a rheostat, fine-tuning aurora B PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19834545 activity in vivo. Materials and methods Cell culture DT40 cells were grown in suspension in RPMI 1640 medium supplemented with 10% FBS, 1% chicken serum, and 100 U/ml penicillin streptomycin and 300 mg/ml L-glutamine and maintained in 5% CO2 at 39C at no more than 106 cells/ml. Doxycycline at a final concentration of 10500 ng/ml was added to the culture medium to repress transcription of the promoter-hijacked endogenous INCENP locus. Gene targeting The incenp1 gene is located at chromosome 5, position 18,267,63 18,291,339. Library screening A DT40 genomic library was screened using a 1.2-kb fragment from the cloned INCENP cDNA. After the third round of screening, one clone, which contained the ATG of incenp1, was used for the construction of the targeting vectors. Promoter hijack vector The promoter hijacktargeting vector was assembled in modified pTre-tight plasmid. The 5 arm and the 3 arm obtained from phage DNA were transferred into the SpeI and PvuIIXbaI sites within the modified pTre-tight, respectively. The resultant targeting vector was linearized using PvuI and transfected into wild-type DT40 clone 18 cells. Targeting events were verified with a 5 probe amplified from phage DNA. After the first targeting, the resistance cassette was excised by transient transfection of a plasmid encoding Cre recombinase. The heterozygote cells were transfected with a kif4a-tTA2 construct before second allele targeting. Disruption vector The disruption vector was based on a pBK-CMV plasmid containing a 4,528-bp incenp genomic DNA-covering region 18,265,04418,269,571. A puromycin resistance cassette was inserted into the SalI site 81 bp downstream from the ATG in the incenp1 locus in the opposite orientation from the incenp open reading frame. The resultant targeting ve