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.

Schizosaccharomyces pombe cells divide by medial fission throughout contraction of an actomyosin ring and deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomoysin ring and septum assembly,little information is available concerning the mechanism of cell separation. Here we describe the characterization of eng1+, a new gene that encodes a protein with detectable endo-β-1,3-glucanase activity and whose deletion is not lethal to the cells but does interfere in their separation. Electron microscopic observation of mutant cells indicated that this defect is mainly due to the failure of the cells to degrade the primary septum, a structure rich in β-1,3-glucans, that separates the two sisters cells. Expression of eng1+ varies during the cell cycle,maximum expression being observed before septation, and the protein localizes to a ring-like structure that surrounds the septum region during cell separation. This suggests that it could also be involved in the cleavage of the cylinder of the cell wall that covers the division septum. The expression of eng1+ during vegetative growth is regulated by a C2H2 zinc-finger protein (encoded by the SPAC6G10.12c ORF), which shows significant sequence similarity to the Saccharomyces cerevisiae ScAce2p,especially in the zinc-finger region. Mutants lacking this transcriptional regulator (which we have named ace2+) show a severe cell separation defect, hyphal growth being observed. Thus, ace2p may regulate the expression of the eng1+ gene together with that of other genes whose products are also involved in cell separation.

ENG1 (YNR067c), a gene encoding a new endo-1,3-␤-glucanase, was cloned by screening a genomic library with a DNA probe obtained by PCR with synthetic oligonucleotides designed according to conserved regions found between yeast exo-1,3-␤-glucanases (Exg1p, Exg2p, and Ssg1p). Eng1p shows strong sequence similarity to the product of the Saccharomyces cerevisiae ACF2 gene, involved in actin assembly "in vitro," and to proteins present in other yeast and fungal species. It is also related to plant glucan-binding elicitor proteins, which trigger the onset of a defense response upon fungal infection. Eng1p and Acf2p/Eng2p are glucan-hydrolyzing proteins that specifically act on 1,3-␤ linkages, with an endolytic mode of action. Eng1p is an extracellular, heavily glycosylated protein, while Acf2p/Eng2p is an intracellular protein with no carbohydrate linked by N-glycosidic bonds. ENG1 transcription fluctuates periodically during the cell cycle; maximal accumulation occurs during the M/G 1 transition and is dependent on the transcription factor Ace2p. Interestingly, eng1 deletion mutants show defects in cell separation, and Eng1p localizes asymmetrically to the daughter side of the septum, suggesting that this protein is involved, together with chitinase, in the dissolution of the motherdaughter septum.The yeast cell wall is a rigid structure that preserves the osmotic integrity of the cell and determines cellular morphology during the different stages of the life cycle. In Saccharomyces cerevisiae, the cell wall is essentially made up of highly mannosylated proteins and three different polysaccharide chains: (i) the predominant, linear 1,3-␤-glucan, (ii) a minor, highly branched 1,6-␤-glucan, and (iii) chitin. All of these components are covalently linked in vivo as part of a macromolecular structure composed of what has been called the "flexible building block," in which mannoproteins are linked to the 1,3-␤-glucan either directly (in the case of PIR proteins) or through a molecule of 1,6-␤-glucan (glycosylphosphatidylinositol proteins) (reviewed in references 8, 43, and 57).Glucans are the main components of the yeast cell wall and are responsible for the rigidity and mechanical strength of this structure. Although they do not undergo appreciable turnover during vegetative growth, it has been proposed that limited site-directed hydrolysis of the rigid skeletal wall ␤-glucans, mediated by endogenous ␤-glucanases, probably takes place during several morphogenetic processes, such as budding, wall growth, conjugation, and ascus formation (8, 42, 58).Two broad classes of 1,3-␤-glucanases occur in yeasts, the exoglucanases and the endoglucanases. As measured by their activity on the substrate laminarin (a linear 1,3-␤-glucan), exo-1,3-␤-glucanases account for the greater part of total glucanase activity in yeasts and hydrolyze the ␤-O-glycosidic linkages at the nonreducing end of the polymer chain, resulting in the release of glucose. These enzymes are not particularly specific because they usually also act on 1,6-␤ linkages...

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Schizosaccharomyces pombe cells divide by medial fission through contraction of an actomyosin ring and deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Here we describe the identification of seven genes (adg1؉ , and mid2 ؉ ) whose expression is induced by the transcription factor Ace2p. The expression of all of these genes varied during the cell cycle, maximum transcription being observed during septation. At least three of these proteins (Eng1p, Agn1p, and Cfh4p) localize to a ring-like structure that surrounds the septum region during cell separation. Deletion of the previously uncharacterized genes was not lethal to the cells, but produced defects or delays in cell separation to different extents. Electron microscopic observation of mutant cells indicated that the most severe defect is found in eng1⌬ agn1⌬ cells, lacking the Eng1p endo-␤-1,3-glucanase and the Agn1p endo-␣-glucanase. The phenotype of this mutant closely resembled that of ace2⌬ mutants, forming branched chains of cells. This suggests that these two proteins are the main activities required for cell separation to be completed.