Schizosaccharomyces pombe Essential Genes: A Pilot Study

Fission Yeast Genes Essential for Vegetative Growth

To estimate the percentage of S. pombe genes required for vegetative growth, we screened a contiguous region of chromosome II containing 100 ORFs. The 253-kb region, selected at random, is likely to be appropriate to estimate the percentage of essential genes. First, according to phylogenetic criteria, the gene distribution within this region containing 100 genes is similar to our estimated distribution for the S. pombe genome (Fig. 1B). Second, assuming that fission yeast genes play a similar role than their budding yeast homologs, the genes appear to be organized randomly relative to their function, with the exception of four genes encoding putative ribosomal proteins (for an estimated total of ∼55 on the entire genome). Interestingly, cytoplasmic ribosomal subunit-encoding genes also appear to cluster in S. cerevisiae (CYGD,http://mips.gsf.de/proj/yeast/CYGD/db/index.html).

We designed 85 pairs of primers for targeted PCR-based gene deletion (Bähler et al. 1998) because deletion of the other 15 genes had already been published (Table1). After transformation of a fission yeast diploid strain with the PCR-amplified deletion cassette, geneticin-resistant clones were selected and the gene deletion was checked by colony PCR (see Methods). Diploids were sporulated and four-spored asci dissected on rich medium. When tetrads did not contain spores that could form geneticin-resistant colonies, the gene deletion was classified as giving a lethal phenotype (Table1). We deleted 65 of the 85 genes by this procedure. Of the 20 remaining genes, the deletion cassette could not be PCR amplified for 3, and correctly deleted geneticin-resistant colonies could not be obtained for the other 17, despite repeating the deletion procedure up to six times. Eight of these undeletable 17 genes were located within a string of 9 contiguous genes that lie between SPBC106.10 andSPBC106.20 ORFs (Table 1), suggesting that this whole chromosomal segment of 18 kb was refractory to gene deletion. We measured the recombination frequency between two markers (cut 4 and cdc13) flanking this segment to check whether the region was a cold spot for recombination. Analysis of random spores generated by a cross between cut4-533 and cdc13-117indicated that the genetic distance between the two markers was 5 cM, compatible with their physical separation of 30 kb, suggesting that this region is not a recombination cold spot. The efficiency with which genes were correctly deleted varied from 5%–100% with an average of 51% based on 650 geneticin-resistant clones analyzed. Including the genes for which the deletion phenotype had already been published, 14 of the 80 genes analyzed were essential for fission yeast vegetative growth (Table 1). This suggests that the percentage of essential genes in S. pombe is 17.5%, compared with 17.8% in budding yeast (YPD; see Garrels 2002), with an interval of confidence (P₉₀) for S. pombe essential genes of 9.5%–25.5%.

Nine of the 14 S. pombe essential genes of Table 1 have been previously described and can be classified into the functional categories of genes described by MIPS (CYGD,http://mips.gsf.de/proj/yeast/CYGD/db/index.html). For the other five fission yeast essential genes, a putative function can be assigned by homology with S. cerevisae. Classification into functional categories reveals that among the 14 S. pombe essential genes of Table 1, 6 belong to the so-called Protein Fate functional category that includes genes involved in protein folding, modification, and targeting. They include stt3, SPBC106.06, cut4, SPBC582.07c SPBC1685.03, and sec61, with cut4, and possiblySPBC106.06 and SPBC582.07c, being also required for completion of mitosis. Another four genes (cdc13, mob1, alp6, cut12) are essential for mitosis. SPBC582.11c is likely to encode the fission yeast homolog of Nup84p nucleoporin; cdt1is required for DNA replication initiation (Nishitani et al. 2000); rhb1 encodes a Rheb-related GTPase that putatively regulates alternative responses to limiting nutrients (Mach et al. 2000); and SPBC1271.13 probably encodes a ribosomal protein.

Essential Genes: Comparison Between S. pombe andS. cerevisiae

We then tested whether deletion of homologous genes in the two yeasts showed the same deletion phenotype (whether essential or not), choosing the closest S. cerevisiae homolog of each of theS. pombe genes from Table 1 and Figure2B (see below). Among the 81 S. pombe genes with an homolog in budding yeast, 88% (71 genes) show the same deletion phenotype in both yeasts, 6% (5 genes) are essential for S. cerevisiae but not for S. pombe, and 6% (5 genes) are essential for S. pombe but not S. cerevisiae (Table 2). This means that of the 15 fission yeast essential genes included in our study, only 10 (67%) are also essential for budding yeast growth. This number is similar to that calculated from previously published data (PombePD), which reveals that 135 of the 198 (68%) S. pombe essential genes with S. cerevisiae homologs are also essential forS. cerevisiae. These data indicate that, although the absolute percentage of essential genes is similar between S. cerevisiaeand S. pombe, surprisingly only two-thirds of the essential genes in one yeast have essential homologous genes in the other yeast.

Figure 2.

Correlation between age and “essentiability” of the S. pombe genes. (A) A simplified phylogenetic tree of the eukaryotic kingdom (Sipiczki 2000) is given. The arrows indicate the point of appearance of S. pombe class Ib, Id, III, and V genes on the tree. (B) Additional S. pombe genes from class Id and class III were deleted using the procedure described in Methods. The phenotype of the deletion is given in the third column. The deletion phenotype of the S. cerevisiae closest homolog of each S. pombe gene is given in the last column. (C) Lethality in gene classes Ib, Id, III, and V of S. pombe andS. cerevisiae is compared. Intervals of confidence (P₉₀) are given for each class of genes.

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One hypothesis would be that, in budding yeast, the genes have been duplicated to compensate for essential gene loss. However, analysis of Table 1 genes reveals that it is unlikely in fission yeast. TheSPBC1271.13 and SPBC582.11c ORFs, which are required for S. pombe growth but do not have essential homologous genes in budding yeast, have not been duplicated in S. cerevisiae. Similarly, the YGR147c, EXO70, and YMR093wORFs of S. cerevisiae, which do not have essential homologs in fission yeast, do not belong to gene families in S. pombe. On the other hand, CCT4, which is required for growth of S. cerevisiae and S. pombe, belongs to gene families in both yeasts. Moreover, most of the paralogous genes of budding yeastCCL4 are also essential, suggesting the existence of essential gene families. Other gene families, like the MFS superfamily of permeases, which includes the nonessential SPBC1271.10c ORF (Table 1), are very unlikely to comprise a high percentage of essential genes, as revealed by S. cerevisiae studies (for review, seeSa-Correia and Tenreiro 2002).

Age of S. pombe Genes and Whether They Are Essential

We then tested whether a gene was essential and correlated it with the time of appearance of a gene on the life tree. Sipiczki (2000) has proposed a consensus tree for eukaryotes based on molecular phylogeny of both 18s rRNA and HMG-CoA reductase sequences. If we refer to this tree shown in Figure 2A, we can postulate that our gene classes appeared in the following order: Ib > Id > III > V. To estimate the percentage of essential genes in each of these classes, we deleted another 24 genes from both class Id and class III (Fig. 2B). Together with the data from Table 1, we estimate that lethality in the classes is as follows: Ib, 25% (6/24); Id, 18% (3/17); III, 7% (1/15), and V, 5% (1/19). From these data, if we consider only the fission yeast genes that do not have homologs within the prokaryotic branch, the more ancient the gene is, the more likely it is to be essential (Fig. 2C). Data from the S. cerevisiae genome (YPD, see Garrels 2002) give a similar profile (Fig. 2C). Focusing on the genes with homologs in both eukaryotic and prokaryotic cells (Table 1, class Ia), we find that 22.5% (4/18) are essential for S. pombe. Data in S. cerevisiae are similar as we estimate that 26% of class Ia genes are required for budding yeast growth. Comparing this data with the average of 17.5% of essential genes for the whole genome, we conclude that ancient genes maintained in all eukaryotic species or in both eukaryotic and prokaryotic species, are more likely to be essential. In contrast, yeast-specific genes (class V), which have appeared recently, are less likely to be essential.

We then focused on S. pombe class IV genes, which have an homolog in the metazoa branch but do not have an homolog in the S. cerevisae lineage, deleting another 36 genes within this class (Table 3). Of these 40 genes, only 1 (cdt1) was found to be essential (Hofmann and Beach 1994). However, in this case, a functionally equivalent gene to cdt1has been reported in S. cerevisiae, which has very low sequence similarity (Tanaka and Diffley 2002). This may be a highly diverged gene derived from a common ancestor or may be an example of nonorthologous gene replacement (Koonin et al. 1996), when a gene is functionally replaced by another that is unrelated by descent. The two genes may, however, share limited sequence similarity acquired by convergent evolution. Assuming that class IV genes are more likely to be the result of gene loss instead of lateral transfer from plants/animals to S. pombe, we conclude that a fission yeast gene for which the homolog has been lost in the budding yeast lineage is very unlikely to be essential, although its origin may be ancient.

Protein Sequence Comparison

S. pombe proteins from PombePD (see Wood et al. 2002) were compared to nr database using BLASTP (Altschul and Lipman 1990) with a cutoff E value of 10⁻⁵.

Gene Deletion

PCR-based gene deletion, using the kan^R marker, was performed according to Bähler et al. (1998) with flanking sequences of 80 bp. An h+/h− ura4-D18/ura4-D18 leu1-32/leu1-32 ade6-M210/ade6-M216 S. pombe diploid strain was transformed with the PCR product, and geneticin-resistant colonies were selected on YES medium containing 100 μg/mL G418 (Life Technologies). Gene deletion was checked by colony PCR. Diploid strains were sporulated and tetrads were dissected as described in http://www.bio.uva.nl/pombe/. The deletion phenotype was assessed on YES medium.