We have sequenced and annotated the genome of ®ssion yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly re¯ecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have signi®cant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identi®ed, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.We report here the completion of the fully annotated genome sequence of the simple eukaryote Schizosaccharomyces pombe, a ®ssion yeast. It becomes the sixth eukaryotic genome to be sequenced, following Saccharomyces cerevisiae 1 , Caenorhabditis elegans 2 , Drosophila melanogaster 3 , Arabidopsis thaliana 4 and Homo sapiens 5,6 . The entire sequence of the unique regions of the three chromosomes is complete, with gaps in the centromeric regions of about 40 kb, and about 260 kb in the telomeric regions. The completion of this sequence, the availability of sophisticated research methodologies, and the expanding community working on S. pombe, will accelerate the use of S. pombe for functional and comparative studies of eukaryotic cell processes.

The minichromosome maintenance (or MCM) protein family is composed of six related proteins that are conserved in all eukaryotes. They were first identified by genetic screens in yeast and subsequently analyzed in other experimental systems using molecular and biochemical methods. Early data led to the identification of MCMs as central players in the initiation of DNA replication. More recent studies have shown that MCM proteins also function in replication elongation, probably as a DNA helicase. This is consistent with structural analysis showing that the proteins interact together in a heterohexameric ring. However, MCMs are strikingly abundant and far exceed the stoichiometry of replication origins; they are widely distributed on unreplicated chromatin. Analysis of mcm mutant phenotypes and interactions with other factors have now implicated the MCM proteins in other chromosome transactions including damage response, transcription, and chromatin structure. These experiments indicate that the MCMs are central players in many aspects of genome stability

The fission yeast Schizosaccharomyces pombe is a popular model system, and has been particularly influential in studies of the cell cycle and chromosome dynamics. Despite its differences from Saccharomyces cerevisiae, the tools and methods for fission yeast are conceptually similar to those used in budding yeast. Here, we present basic methods sufficient for a beginner in this system to carry out most required manipulations for genetic analysis or molecular biology.

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(2). The other regulated system was constructed from the tetracycline-inducible system described by Faryar and Gatz (3). The two constitutive promoters were the previously described vectors pART1 (containing the adh promoter; .(4) and pSMl (containing the SV40 promoter; 5).The expression vector REP3, containing the thiamine-inducible nmtl promoter (1), and its derivatives REP41 and REP81 (2), which have lower levels of activity due to mutation, all contain an ATG within their polylinker. This was destroyed by insertion of a Xho linker; these derivatives are called REP3X (full strength nmtl), REP41X (slightly weaker; nmtl*) and REP81X (much weaker; nmtl **). The tetracycline expression system was originally described in (3) and modified for more general use. The expression vector, pSLF101, has the constitutive CaMV promoter adjacent to the tet operator as described in (3); in addition, it contains a polylinker, and the LEU2 and arsI markers for selection in fission yeast. A ura4+ marked version, pSLF102, was also constructed but not tested. The tet represser, which blocks transcription from the otherwise constitutive CaMV promoter, was provided by integration of plasmid pSLF104 into a strain of genotype h-ade6-704 leul-32 ura4-D18. The plasmid contains the marker sup3-5, which suppresses ade6-704, and the tet represser gene under control of the S.pombe adh promoter, as described by Faryar and Gatz (3). The resulting fission yeast strain, FY191, thus produces a constitutive tetracycline repressor protein.A 3 kb BamHI cassette containing the beta-galactosidase gene (6) was cloned into the BamHI site in the polylinkers of all the vectors, and transformed into a fission yeast strain of genotype h-leul-32 ura4-D18. Media and methods were as described in (7). The tet-inducible pSLF101-lacZ was also transformed into the strain with the integrated tetracycline repressor. Betagalactosidase assays were carried out as described in (8). All assays were carried out after growth for 24 hrs at 30°C on strains grown to