GATGTCGCCTCCACCAGC-39) and reverse (59- GCGCTGCAGAGGGATCTGTCTAGCAGCTG-39) primers and ligated into pEGFP-N1 (Clontech) at the XhoI and PstI sites. pHybLex/Zeo-SpErh1p. The Sperh1+ CDS was amplified from pUC18/cSperh1 with forward (59-GCGGAATTCTCGCCTC- CACCAGCAGAATC-39) and reverse (59-GCGCTCGAGT- TAAGGGATCTGTCTAGCAGC-39) primers and ligated into pHybLex/Zeo (Invitrogen) at the EcoRI and XhoI sites. pEGFP-N1/ER, pmCherry-N1/Ciz1, pHybLex/Zeo-ER, pYESTrp2/Ciz1 and pYESTrp2/PDIP46 were described pre- viously [9,11]. Disruption of the Sperh1+ Gene A full-length MX6-type disruption cassette of 1924 bp was amplified by PCR from pFA6a-59arm(Sperh1)-Kan-39arm (Sperh1) with forward (59-TCGCTACTCAATAACTGTGC- TAC-39) and reverse (59-AGACAAATGCCATTTATTA- GAAC-39) primers and introduced into S. pombe cells (strains ZBM1004, FY7269 and FY7266) by chemical transformation. After overnight incubation in YES, transformants were selected on YES agar supplemented with Geneticin (G418 sulfate, Gibco BRL, 150 mg/ml) and verified by PCR using total DNA and forward (59-TAAATTGTGAGAACTGCAAAG-39) and reverse (59-GGAATTGGTCCTTTGTCTTGC-39) primers that anneal to the genomic DNA outside of the 59 and 39 arms (for the presence of the 2074-bp PCR product). Epitope Tagging of the Sperh1+ Gene in its Chromosomal Locus Sperh1 was chromosomally tagged with yEGFP in strain FY7269 by employing pFA6a-39ORF(Sperh1)-link-yEGFP-Kan-39arm (Sperh1) as a template, forward (59-AGGAATCTATT- GATGTCTCAA-39) and reverse (59-AGACAAATGCCATT- TATTAGAAC-39) primers and the same strategy as used for the gene disruption. Microscopy Images of yeast cells were acquired on a Nikon Eclipse TE200 microscope with Hoffman Modulation Contrast, HMC LWD 406/0.55 objective and DS-Fi1 CCD camera head or a Nikon Eclipse Ti-E fluorescence microscope with Nomarski Interference Contrast, Apo TIRF 1006/1.49 Oil DIC objective and DS-5Mc CCD camera head. For staining with DAPI, 2.5 ml of yeast cell References 1. Wojcik E, Murphy AM, Fares H, Dang-Vu K, Tsubota SI (1994) Enhancer of rudimentary p1, e(r)p1, a highly conserved enhancer of the rudimentary gene. Genetics 138: 1163–1170. 2. Isomura M, Okui K, Fujiwara T, Shin S, Nakamura Y (1996) Cloning and mapping of a novel human cDNA homologous to DROER, the enhancer of the Drosophila melanogaster rudimentary gene. Genomics 32: 125–127. 3. Gelsthorpe M, Pulumati M, McCallum C, Dang-Vu K, Tsubota SI (1997) The putative cell cycle gene, enhancer of rudimentary, encodes a highly conserved protein found in plants and animals. Gene 186: 189–195. 4. 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ERH Genes from Fission Yeasts suspension in water was spread on a microscope slide, fixed on a heating plate at 65uC for a few seconds (until water just evaporated) and 2.5 ml mounting medium with DAPI (UltraCruz, Santa Cruz) was added followed by covering with a 16 mm square coverslip. Images of HeLa cells expressing fluorescent proteins were acquired on a Zeiss LSM510 confocal microscope as described previously [9,11]. Flow Cytometry Yeast cells were stained with propidium iodide (PI) according to [25,50]. Briefly, after fixing in 70% ethanol cells were washed with 50 mM sodium citrate, pH 7.0, suspended in the same buffer supplemented with ribonuclease A (100 mg/ml) and incubated for 2 hours at 37uC followed by adding an equal volume of the same buffer supplemented with PI (final concentration of 4 mg/ml) and short sonication bursts. Cells were sorted based on DNA content using a Becton-Dickinson FACSCalibur flow cytometer and cell cycle analysis was performed with the CellQuest Pro software. Other Methods Maintenance and transfection of HeLa cells (ECACC 93021013), yeast two-hybrid analysis and protein identification by tandem mass spectrometry were performed as described previously [9,11]. Acknowledgments We thank YGRC, NIG, DSMZ and CBS for providing the fission yeast strains. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco. We are also grateful to Drs. M. Dadlez and J. Debski from the Laboratory of Mass Spectrometry (Institute of Biochemistry and Biophysics, PAS, Poland) for the mass spectrometry analysis, Dr. B. Paterczyk from the Laboratory of Electron and Confocal Microscopy (this institution) and Dr. I. 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