Six-way Venn diagram showing the number of conserved loci identified among the different fungi. Six-frame translations of six different fungi (key, top left) were each compared to that of S. pombe. Each shape represents the set of loci in S. pombe with significant conservation to one of the six other fungi. Intersections between shapes represent intersections between each of these sets, and the size of each intersection is indicated. For example, 1921 conserved loci were identified between S. pombe and S. octosporus (top center), and an additional 496 loci between S. pombe, S. octosporus, and S. japonicus. (A) All conserved loci. (B) Loci after filtering to remove known coding regions.

Tetrad analysis of knockout strains. Asci from the appropriate new∷nat/new⁺ ade6.M210/ade6.M216 h⁺/h− diploid strains were dissected on rich yeast extract supplemented (YES) medium at 25°. Images of colony formation on the left demonstrate the essentiality of new21⁺ and the slow-growth phenotypes of new1∷nat, tam7∷nat, and new8∷nat cells. The bright-field microscopy images show increased magnification of new/tam⁺ (middle) and newx∷nat/tamx∷nat (right) colonies (denoted as “Wild type” and “Deletion”). The far right panel of the new8∷nat cells shows the inability of the microcolonies formed at 25° to grow 4 days after restreaking onto fresh YES at 25°.

Spot-test analysis reveals the temperature-dependent lethality of new1∷nat and tam7∷nat. Cells from cultures of the indicated strains grown to mid-log phase in rich YES liquid medium were plated in fivefold serial dilutions so that the final dilution plated around five cells on YES plates that were then incubated at the indicated temperatures. The limited ability of the plated new1∷nat cells to grow and divide at 32° is clear from a comparison of the magnified images, highlighting growth of the same strain on a YES plate at 25° (top right) and 32° (bottom right).

Phenotypic characterization of knockout strains. (A–E) Calcofluor DAPI staining of the indicated strains. S. pombe cells grow by linear extension until they reach a critical size threshold. Nuclear division is then initiated, followed by the contraction of the cytokinetic F-actin ring, resulting in separation of the two cytoplasms of the incipient daughter cells. F-actin ring contraction is coupled with the deposition of the cell-wall material of the primary septum that stains strongly as a white bar across the cell equator between the separated nuclei (Marks and Hyams 1985). This primary septum is subsequently degraded to separate the two daughter cells. Thus, the length of cells with the transecting bright calcofluor-positive stain is a direct indication of the timing at which cells commit to mitosis (Nurse 1975). It is clear from B–D that the cells with this bright bar in strains new5.Δ, tam11.Δ, and tam7.Δ are longer than the wild-type controls in A and so are delayed in division. (E) The accumulation of excessive regions of calcofluor staining in new1.Δ cells is indicative of severe defects in septation in some cells in the culture. (F) Anti-tubulin, anti-Sad1 immunofluorescence of new1.Δ cells. DAPI staining of chromatin either alone or in combination with differential interference contrast imaging visualizes the position of the chromatin relative to the cell periphery, as indicated on each panel in the figure. A range of mitotic defects was apparent, including, most notably, a failure in chromosome segregation along elongating spindles (cell 1) and the formation of monopolar spindles with expansive arrays of red microtubules extending from single green foci of Sad1 staining [cell 2: F(II)]. Highly condensed, unsegregated chromosomes cluster around these Sad1 foci [cell 2: F(IV)]. F(IV) and F(IV′) show the same DAPI images of chromatin merged with two different focal planes in the Sad1 channel because the different spindle pole bodies (SPBs) in cells 1 and 2 reside in different focal planes.

Spot-test analysis demonstrates complementation of the compromised growth phenotype of new1∷nat, new5∷nat, new8∷nat, and tam7∷nat following exogenous provision of mRNA encoding the appropriate ORF. Cells from cultures of the indicated strains were grown to mid-log phase in rich YES liquid medium containing 100 μg·ml⁻¹ hygromycin to select for the presence of the plasmid and plated in fivefold serial dilutions, so that the final dilution plated around five cells on YES plates that were then incubated at 25°. The provision of the appropriate reading frame rescued the slow-growth phenotype in each case. The subtlety of the growth defect in new5∷nat meant that the differential colony size relative to wild-type controls is only transiently visible in spot-test analyses (compare the clear complementation here with the apparent wild-type growth in new5∷nat cells in Figure 3); however, it is clear that, even in this case, the exogenous provision of the appropriate mRNA complements the slow growth arising from deletion of this gene.

A cartoon representing the gene detection pipeline. The entire genome is translated in all three forward and reverse reading frames and segmented at putative stop codons. Data from different sources are compared to these translations and mapped back to the genome. Matching features that do not overlap with existing genes are used to infer novel genes and/or to refine gene structures. (1) MS/MS data searched against all putative protein sequences that could be expressed from the genome identify novel protein-coding genes (2) and refine 5′ and 3′ ends of others (3). MS/MS data matching pseudogenes, and genes annotated as noncoding, confirm translation and lead to altered annotation (4). Pfam searches against six-frame translations identify additional protein domains (5). Comparative genomic searches between whole-genome protein translations of six fungal species to S. pombe are used to infer novel protein coding genes (6).

Examples of loci identified by the pipeline and confirmed by 5′ and 3′ RACE. Part of the S. pombe genome represented by the X:Map genome browser (http://xmap.picr.man.ac.uk) is shown. The chromosome runs across the middle of the figure with genes on the forward strand shown above the center line and those on the reverse strand, below. Boxes represent genes, with the transcript structure and exons indicated by the smaller boxes within. (A) The 5′ extension to SPBC16H512c identified by two novel peptides. The original and revised gene structures are shown; the revised gene structure incorporates a locus previously annotated as a ncRNA (noncoding RNA). (B) Location of new10, enhancer of rudimentary homolog-like protein, identified by comparative genomics.