Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe
Dong-Uk Kim1,14, Jacqueline Hayles2,14, Dongsup Kim3,14, Valerie Wood2,
4,14, Han-Oh Park5,14, Misun Won1,14, Hyang-Sook Yoo1,14, Trevor Duhig2, Miyoung Nam1, Georgia Palmer2, Sangjo Han3, Linda Jeffery2, Seung-Tae Baek1, Hyemi Lee1, Young Sam Shim1, Minho Lee3, Lila Kim1, Kyung-Sun Heo1, Eun Joo Noh1, Ah-Reum Lee1, Young-Joo Jang1, Kyung-Sook Chung1, Shin-Jung Choi1, Jo-Young Park1, Youngwoo Park1, Hwan Mook Kim6, Song-Kyu Park6, Hae-Joon Park5, Eun-Jung Kang5, Hyong Bai Kim7, Hyun-Sam Kang8, Hee-Moon Park9, Kyunghoon Kim10, Kiwon Song11, Kyung Bin Song12, Paul Nurse2,13 & Kwang-Lae Hoe1,6
We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast—the only other eukaryote for which a comprehensive knockout library exists—revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth. Systematic genome-wide gene deletion collections of eukaryotic organisms provide powerful tools for investigating molecular mechanisms in basic biology and for identifying pathways that can be targeted in bioengineering or medical applications, as shown by pioneering studies with the budding yeast Saccharomyces cerevisiae1–5. The construction of systematic gene deletion collections is difficult, although RNA interference (RNAi) provides a popular alternative approach to ablate gene activity in many organisms. However, RNAi approaches suffer from drawbacks such as partial knockdown of gene expression and off-target effects. For example, RNAi screens in fly and human cells revealed only a 10–38% overlap in genes identified as being required for the cell cycle between these two organisms6. We have constructed a genome-wide gene deletion set for the fission yeast Schizosaccharomyces pombe. Fission and budding yeast are not closely related and differ in a number of aspects including organization of the cell cycle, heterochromatin, complexity of centromeres and DNA replication origins and the prevalence of introns7, which makes their comparison valuable for defining genes and processes required more generally in eukaryotes. Here, we have identified similarities and differences in gene dispensability between the two yeasts and have used growth fitness profiling to identify genes haploinsufficient or haploproficient for growth. RESULTS Deletion construction and gene dispensability We have constructed 4,836 heterozygous deletions covering 98.4% of the 4,914 protein coding open reading frames (ORFs) based on the annotated genome sequence7 (http://www.genedb.org/genedb/ pombe, 01/04/08) (Online Methods and Supplementary Table 1; for all the PCR primer sets and the mapping data, see Supplementary Data 1 and 2; also available at http://pombe.kaist.ac.kr/nbtsupp/). In addition, we have deleted 9 Tf2 transposons, 39 dubious genes8 and 48 pseudogenes (Supplementary Table 2). Each gene was deleted and replaced using homologous recombination by a ‘deletion cassette’ containing the KanMX marker gene9 (Supplementary Data 3) flanked by a pair of unique molecular bar codes (Fig. 1a, Supplementary Fig. 1 and Supplementary Table 1). Several pilot scale deletion studies have been carried out10–12 and it was suggested that 40~80 bp of homology is not always sufficient for the recombination required for the systematic deletion of genes in fission yeast12. Both block PCR
? 2010 Nature America, Inc. All rights reserved.
Omics Research Centre, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong, Daejeon, Korea. 2Cancer Research UK, The London Research Institute, London, UK. 3Department of Bio and Brain Engineering, Korea Advanced Institute of Science & Technology (KAIST), Yuseong, Daejeon, Korea. 4Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. 5Bioneer Corp., Daedeok, Daejeon, Korea. 6Bioevaluation Centre, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Chungcheongbuk-do, Korea. 7Department of Bioinformatics & Biotechnology, Korea University, Jochiwon, Chungnam, Korea. 8School of Biological Sciences, Seoul National University, Seoul, Korea. 9Department of Microbiology, Chungnam National University, Yuseong, Daejeon, Korea. 10Division of Life Sciences, Kangwon National University, Chuncheon, Kangwon-do, Korea. 11Department of Biochemistry, Yonsei University, Seoul, Korea. 12Department of Food and Nutrition, Chungnam National University, Yuseong, Daejeon, Korea. 13The Rockefeller University, New York, New York, USA. 14These authors contributed equally to this work. Correspondence and requests for material should be addressed to K.-L.H. (firstname.lastname@example.org). Received 6 January; accepted 30 March; published online 16 May 2010; doi:10.1038/nbt.1628
nature biotechnology? VOLUME 28
Figure 1 Deletion construction and gene dispensability. (a) Gene deletion cassette containing the KanMX4 gene flanked by unique bar codes (UPTAG/DNTAG) and regions of homology to the gene of interest (RHG). The cassette replaced the ORF of interest by homologous recombination at the RHG regions. (b) Construction of deletion mutants. All 4,836 protein coding genes were deleted using serial extension PCR (31.3%), block PCR (63.2%) or total gene synthesis (5.4%). The remaining 78 genes could not be confirmed as deleted owing to ambiguous sequencing results, recombination failure or inviability of the heterozygous diploids. (c) Dispensability of 4,836 protein coding genes. For 3,626 (2,729 + 897) genes the dispensability was previously unknown. ND, not done.
Gene b synthesis (263)
Not made (78)
Nonessential (this study) 2,729 Essential (this study) 897
and total gene synthesis methods13 were developed to overcome this problem by increasing the length of homology from ~80 bp to ~350 bp (Fig. 1b and Supplementary Figs. 2–4). We confirmed that the deletion mutants were correctly replaced with the KanMX marker using PCR and dideoxy sequencing (Supplementary Fig. 5). For some genes constraints on primer selection for block PCR resulted in <100% of the ORF being deleted (for the amount of ORF deleted see Supplementary Table 1, column C KO%). Of the 4,836 genes deleted, at least 4,328 genes (87.6%) have >80% of their ORFs removed. In addition we carried out Southern blot analysis to determine the frequency with which the deletion cassette integrated elsewhere in the genome and estimated it to be <1% (Supplementary Fig. 6). We determined the essentiality of 4,836 genes by sporulating each heterozygous deletion diploid strain and then observing the germinating haploid spores microscopically. Essentiality was confirmed by tetrad analysis for all genes initially characterized as essential. We found that 26.1% of fission yeast genes (1,260/4,836) were essential and 73.9% (3,576/4,836) were nonessential for viability of haploid cells in the growth conditions we used. This analysis determined the dispensability for 3,626 genes that had not been deleted previously (Fig. 1c). Comparisons with published data for 1,210 genes revealed that the dispensability data for 98.4% of our deletions are similar or our data are more likely to be correct, leaving 1.6% as the maximum estimate of the error rate in our study (Supplementary Table 3). These results contrast with budding yeast where 17.8% (1,033/5,776) of genes are essential for viability (http://www.yeastgenome.org/). Fission yeast therefore has 227 more essential genes (1,260–1,033) than budding yeast despite having fewer genes in total (4,836 versus 5,776). Fission yeast has fewer duplicated genes than budding yeast14,15. It is therefore possible that there are more essential genes in fission yeast, because duplication in budding yeast is masking potential essentiality. We examined this possibility by identifying all of the essential genes for each organism with duplications in the other organism (orthologous relationships Sp|Sc, one|many, many|one and many|many). We then identified the cases where these essential genes had orthologs in the other organism that were both nonessential and duplicated (Supplementary Table 1 and Online Methods). This revealed only 67 essential genes in fission yeast and 32 essential genes in budding yeast where essentiality of the orthologs in the other organism could be masked by redundancy. Thus redundancy could account for maximally only 35 (67–32) of the 227 extra essential genes in fission yeast. We conclude that redundancy is not the major reason for the additional essential genes in fission yeast. Analysis of gene dispensability Essential and nonessential genes are distributed evenly throughout the fission yeast genome except within 100 kb of the telomeres on chromosomes 1 and 2 (Fig. 2a). As in other organisms4 genes in the subtelomeric regions showed low essentiality (1.2%) compared to a
Serial PCR (1,515)
Block PCR (3,058)
Nonessential (known) 847
Essential ND (known) 363 78
? 2010 Nature America, Inc. All rights reserved.
genome average of 26.1%. These regions are enriched for paralogs (68.7%, 79/115) and we have shown that duplicated genes are less likely to be essential than single-copy genes (Supplementary Table 4). These regions are also enriched for nonessential species-specific genes related to meiosis and the response to nitrogen starvation, which are less likely to be essential under these assay conditions15. In fission yeast ~46% of genes have introns7 and we found that the essentiality of genes with one or more introns is significantly higher than genes lacking introns (33% versus 21%, P < 10?14) (Fig. 2b). One possible explanation is that essential genes are less likely to be rapidly regulated given that it has already been shown that rapidly regulated stress response–related genes are less likely to contain introns16. Alternatively if introns arose early during eukaryotic evolution, this may be reflected as a bias toward intron-containing essential genes because essential genes are more likely to be ancient than nonessential genes12. The relationship between our gene essentiality data and previously published ORFeome localization17 was also analyzed for ten different cellular locations (Fig. 2c). As in budding yeast3,18 we found that the greatest percentage of essential gene products was localized to the nucleolus, nuclear envelope and the spindle pole body. As previously shown for budding yeast4, essential genes in fission yeast were more likely to be unique, with 93.1% of essential genes (1,173/1,260) being present in single copy compared to 73.9% of nonessential genes (2,643/3,576). In contrast nonessential genes were more likely to be duplicated or species-specific. Comparison of Gene Ontology (GO) term enrichment between the two yeasts revealed that the essential gene sets for both yeasts were significantly (P < 10?2) enriched for core cellular processes, such as macromolecular (DNA, RNA, protein and lipid) metabolism and cellular biosynthesis (transcription initiation and/or translation and ribosome assembly) (Fig. 2d and Supplementary Table 5). In contrast, nonessential genes were significantly (P < 10?2) enriched for regulatory functions (control of gene expression and cell communication) (Fig. 2d and Supplementary Table 6). Nonessential genes were also enriched for conditional or life-cycle specific processes, such as stress response, transmembrane transport and meiosis or sexual reproduction, together with processes that are less likely to be essential in the rich medium and mitotic growth used in our assay conditions. Genes of unknown function were also highly enriched (93%) in the nonessential genes. We predict that many of these genes are involved in biological regulation or condition-specific processes and are not directly involved in primary processes.
VOLUME 28 NUMBER 6 JUNE 2010 nature biotechnology
Chr 1 Cen1 (5.6 M)
Biological process Cellular component org. Cellular macromol. biosyn. Enriched essential Nucleotide/nucleic acid met. RNA processing Ribosome biogenesis Protein localization Translation Mitotic cell cycle DNA replication General transcription Enriched nonessential Unknown Meiotic cell cycle Response to stress Transmembrane transport Cell communication Reg. of gene expression 0 500 1,000 Number of genes 1,500 2,000
Essential genes (%)
Cytoplasm Nucleus Mitochondrion ER Cell periphery Nucleolus Golgi Spindle pole body Nuclear envelope Microtubule 0 5 10 15 20 25 30 35 40 45 Essential genes (%)
Essential (Fission/budding) Nonessential
30 25 20 15 10 5 0 0 1 2 3 >4 Number of introns
? 2010 Nature America, Inc. All rights reserved.
Figure 2 Analysis of gene dispensability. (a) Chromosome distribution of gene dispensability. Essential genes (tall bars) and nonessential genes (short bars) are distributed randomly throughout the genome except within 100 kb of the telomeres (gray boxes), where nonessential genes are enriched. Upper bars represent genes transcribed left to right and lower bars represent genes transcribed right to left. Filled circles in orange represent centromeres. (b) Percentage of essential genes versus number of introns. Percentage of essential genes was plotted against the number of introns within genes. In fission yeast, the percentage of essential genes containing introns is significantly (P < 10?14) higher than the percentage of those lacking introns. The dotted line represents the average percentage of essential genes in the total gene set (26.1%). (c) Percentage of essential genes versus ORFeome localization. The percentage of essential genes was plotted against ten different cellular locations in fission yeast. The dotted line represents the average percentage of essential genes for the total gene set (26.1%). The number of essential gene products localized to the nucleolus, spindle pole body and nuclear envelope is higher than average. The number of essential genes compared to the total for each location is: (i) cytoplasm 564/2,113; (ii) nucleus 601/2,068; (iii) mitochondrion 128/450; (iv) ER 98/436; (v) cell periphery 55/326; (vi) nucleolus 89/217; (vii) Golgi 27/224; (viii) spindle pole body 69/181; (ix) nuclear envelope 29/76; and (x) microtubule 20/71. (d) Comparison of GO analyses of fission yeast and budding yeast genes. Bar chart shows a selection of broad, biologically informative GO terms significantly (P ≤ 0.01) enriched for essential and nonessential genes in fission yeast and budding yeast. For the complete list of processes and for methods used to extract these data, see Supplementary Tables 5 and 6.
Species distribution of essential genes The dispensability profiles for the 4,836-deletion gene set were classified by their gene copy numbers according to their relationship with budding yeast genes (Supplementary Table 4 and x axis in Fig. 3) and into five categories by their species distribution (Supplementary
Essential Fission yeast specific Variable phyla Species distribution Nonessential
Table 4 and y axis in Fig. 3). In a comparison of the entire deletion gene set (4,836) there are 2,841 single-copy genes (n = 1, m ≥1) (n and m are gene copy number in fission yeast and budding yeast, respectively), 855 duplicated genes in fission yeast that are conserved in budding yeast (n > 1, m ≥1) and 1,140 genes found in fission yeast but not conserved in budding yeast (n ≥ 1, m = 0). The 1,260 essential genes were distributed across species as follows: (i) 883 genes conserved only in eukaryotes including humans, (ii) 207 conserved in both bacteria and eukaryotes, including humans, (iii) 91 genes found only in fungi, (iv) 39 genes found with a variable distribution throughout the phyla and (v) 40 fission yeast–specific genes. Essential genes were more likely than nonessential genes to be single copy and to be conserved broadly across species. Of the 1,260 essential fission yeast genes, 1,173 were single copy and only 87 have duplicates (Supplementary Tables 1 and 4). From the total of 974 (883 + 91) essential genes found only
Figure 3 Comparative analysis of gene dispensability profiles of fission yeast. Gene dispensability profiles of 4,836 deletion mutants by gene copy number of fission yeast orthologs compared to budding yeast (x axis) and species distribution (y axis). Compared to budding yeast, fission yeast genes consist of 2,841 single-copy genes (n = 1, m ≥1), 855 duplicated genes (n > 1, m ≥1) and 1,140 genes found in fission yeast but not in budding yeast (n ≥ 1, m = 0), where ‘n’ is the number of genes in fission yeast and ‘m’ is the number of genes in budding yeast. The term ‘eukaryotes’ includes human and the term ‘variable phyla’ includes plants. The area of each circle represents the numbers of genes, where essential and nonessential genes are represented by yellow and blue, respectively.
Eukaryotes + bacteria
n>1 m≥1 Gene copy number
nature biotechnology? VOLUME 28
in eukaryotes, 59 are probably related to genes found in Archaea (Supplementary Table 7). The remaining 915 genes (72.6% of all essential genes) are likely to have arisen within the eukaryotic lineage. This implies that many essential novel gene functions arose with the evolution of the eukaryotic cell. The fidelity of cell division in ancestral unicellular eukaryotes may have been very low, which could be tolerated in evolutionary terms as long as there was overall population growth. However, a multicellular eukaryote requires greater fidelity at each cell division than a unicellular eukaryote, because even moderate levels of random cell death would lead to poor survival of a multicellular organism. It has been estimated that it took around 500 million years for multicellular organisms to arise from an ancestral unicellular eukaryote19, and we propose that during this period there was considerable genomic innovation to generate a unicellular eukaryote with sufficient fidelity at cell division to allow the evolution of multicellularity. Essential genes broadly conserved both in bacteria and eukaryotes were significantly (207 genes, P < 10?2) enriched for respiratory function and primary metabolism of low molecular weight molecules, such as nucleotide or glucose metabolism (Supplementary Table 8). Of 445 fission yeast–specific genes, only 40 were essential for viability (Supplementary Table 9). Some of these genes are implicated in aspects of mitotic and meiotic chromosome segregation10 and such species-specific genes may have played a role in speciation by reinforcing reproductive isolation20. As the majority of essential genes are broadly conserved, it is possible that distant orthologs exist in other eukaryotes, including budding yeast, if some of these apparently species-specific genes are rapidly diverging. To investigate this possibility we re-interrogated the nonconserved essential genes from both yeasts using the same criteria used to build the manual ortholog data set, but relaxing thresholds for candidates to generate seed alignments and building alignments starting from the budding yeast genes rather than the fission yeast genes. This revealed a further four potential orthologs (Supplementary Table 10). This indicates that more in-depth comparisons of the essential nonconserved gene sets may reveal further distant evolutionary relationships and functions. Dispensability comparison of orthologous pairs from the two yeasts Access to deletion collections for both fission yeast and budding yeast allows a robust comparative analysis of dispensability between two evolutionarily distant eukaryotic organisms. To eliminate any complications due to functionally redundant paralogous genes, 2,438 single-copy orthologous pairs (one to ones) for which deletion data are available in both organisms were used for this analysis (Supplementary Table 11). Overall 83% of these genes (2,027/2,438) had the same dispensability in both yeasts (Fig. 4a), suggesting that conserved orthologs in other organisms may also have conserved dispensability. GO enrichment of the conserved one-to-one essential genes in fission and budding yeasts was similar to that of all essential genes (compare Supplementary Table 12 with Supplementary Table 5), whereas the nonessential one-to-one pairs (compare Supplementary Table 13 with Supplementary Table 6) were enriched for additional GO terms, such as DNA damage, Golgi and/or endoplasmic reticulum (ER)-related processes and catabolic processes. As conserved genes can be expected to be under positive selection, these single-copy nonessential genes are likely to contribute to overall cell fitness. For example, the inability to repair nonlethal DNA damage will reduce cell fitness. It is also likely that some processes still take place in the absence of certain components, albeit less efficiently, because of flexibility and plasticity in the processes concerned21. The Golgi/ER-related processes may be complemented by different but related membrane trafficking pathways or components substituting one for the other. The remaining 17% of orthologous pairs (411/2,438) differ in essentiality between the two yeasts; of these, 268 are essential only in fission yeast and 143 are essential only in budding yeast (Fig. 4a). Therefore, there are 125 extra essential genes (268–143) in fission yeast
Mitochondrial translation Other mitochondrial function Iron-sulphur cluster assembly Other processes Spindle/kinetochore associated DNA recombination/repair DNA replication checkpoint Mitotic/SIN signaling Glycosylation/ER associated V-type ATPase Proteosome/ubiquitin associated SUMOylation associated Neddylation associated Proteolysis (peptidases) Met/Thr/Glu metabolism Tryptophan metabolism Ergosterol metabolism Other amino acid metabolism Heme metabolism Purine/pyrimidine metabolism Tubulin-specific chaperone Actin cytoskeleton related
? 2010 Nature America, Inc. All rights reserved.
Essential Nonessential E:NE 268 (11%)
NE:NE 1,312 (54%)
E:E 715 (29%)
NE:E 143 (6%)
Number of genes with different dispensability
Number of genes with different dispensability
Figure 4 Dispensability comparison of orthologous pairs from the two yeasts. (a) Essentiality of nonredundant 2,438 orthologous pairs were compared between the two yeasts. Eighty-three percent of orthologs show conserved dispensability and the remaining 17% show different dispensability. E, essential; NE, nonessential. (b) Functional distribution of orthologs with different dispensability. The 17% of the orthologous pairs with different dispensability were allocated to one of 31 biological terms, 22 of which are shown here. For the complete list of processes and genes, see Supplementary Table 14. Note that genes annotated to mitochondrial functions, certain amino acid metabolic pathways and protein degradation pathways such as neddylation and sumoylation are mostly essential in one yeast and nonessential in the other yeast, whereas other categories show essential genes (although the specific genes are different) in both yeasts under the conditions used in this study. Because there are some differences in the constituents of the standard rich media used for each organism, it is possible that in a few cases different dispensability between the two organisms are due to these differences.
compared to budding yeast in this category, making the difference in dispensability of one-to-one orthologs a major reason why overall there are 227 more essential genes in fission yeast than in budding yeast. To analyze these differences further we identified a set of broad biological processes that encompass the entire set and sorted each gene pair into the most biologically relevant group (Fig. 4b and Supplementary Table 14). The most striking difference was for mitochondrial function (95 orthologous pairs). Of these 89 genes were essential in fission yeast and only six genes in budding yeast. Many of these genes encode components of the mitochondrial translation machinery (69 genes), which is required for mitochondrial DNA (mtDNA) stability. Loss of mtDNA is lethal in fission yeast but not in budding yeast where ‘petite’ mutants lacking mtDNA are viable and mitochondrial translation is not essential22. Conversely, the DNA replication checkpoint genes rad3, rad26 and cds1 are nonessential during normal growth, whereas their respective budding yeast orthologs are essential because of the requirement for degradation of a ribonucleotide reductase inhibitor23. This inhibitor can be degraded by a second checkpoint-independent pathway in fission yeast24,25 and other eukaryotes but not in budding yeast. Other examples of differential essentiality include the biological processes relating to RNA processing and/or export pathways, Golgi/ER transport, spindle/kinetochore/centromere, transcription and/or other chromatin-associated and glycosylation and/or other ER-associated processes. These differences may reflect dissimilarities in the numbers of introns7, centromere structure7, the organization of the Golgi network26,27 and membrane trafficking. Although 83% of the orthologous pairs have conserved dispensability, different essentiality of specific biological processes and defined complexes in 17% of gene pairs may represent life-style differences between these distantly related yeasts. Growth profiling of diploids All fission deletion mutants constructed in this study have been barcoded (Supplementary Table 1), enabling the strains to be examined as an entire set in pooled experiments. Parallel analysis for changes in the growth rate of heterozygous deletion diploid strains has been used in budding yeast to identify potentially rate-limiting steps for cellular growth2,28,29. Using a similar methodology30 (Online Methods and Supplementary Figs. 7–9), we examined the growth rates in yeast extract medium for 4,334 fission yeast heterozygous deletion diploids (Supplementary Table 15; for the microarray raw data see Supplementary Data 4 and 5) and we further examined the growth rate of the 10 slowest haploinsufficient mutants as a proof-of-principle experiment (Supplementary Fig. 10). The growth rates of these ten mutants were found to be comparable to the relative fitness results from the microarray parallel analysis. Comparisons were also made for the haploinsufficient (slower growth) and haploproficient (faster growth) genes in fission yeast and budding yeast (Fig. 5). There were considerably more haploinsufficient genes in fission yeast compared to budding yeast (455 versus 356) when using a growth rate cut-off of <0.97 (Fig. 5 and Supplementary Table 16), whereas there were a similar number of haploproficient genes in both yeasts. The budding yeast life cycle is predominantly diploid and so reduced expression of potentially haploinsufficient genes in diploid cells is likely to have been subject to strong negative selection; this would not be the case for the predominantly haploid fission yeast. To make a more direct comparison between the fission and budding yeasts, we compared the fastest 3% of haploproficient genes (136 versus 183) and the slowest 3% of haploinsufficient genes (138 versus 184) from each organism (Table 1 and Supplementary Table 17). In fission yeast the haploproficient gene set showed GO enrichment for
nature biotechnology? VOLUME 28 NUMBER 6 JUNE 2010
1,500 100 Number of genes 50 0 Fission yeast
0 0.88 1,500
Number of genes
? 2010 Nature America, Inc. All rights reserved.
0.91 0.94 0.97 1.00 Relative growth rate in rich media
Figure 5 A comparison of the relative growth rates for the total set of heterozygous deletion diploids in fission yeast (4,334 genes) and budding yeast (5,921 genes). In fission yeast there are more haploinsufficient genes with a relative growth rate of <0.97 compared to budding yeast (455 versus 356), as shown in the expanded region 0.88–0.97 (Supplementary Table 16).
macromolecule biosynthesis (P < 2.1 × 10?19) particularly ribosomal proteins (Table 1a and Supplementary Table 18). The TOR pathway genes (tor2, tsc1 and mip1) and genes encoding Rab-GTPase activating proteins were also found to be enriched in the haploproficient gene set. The loss of heterozygosity in TSC131, RAB-GTPases32 and also certain ribosomal proteins33 has been implicated in certain human cancers. None of these haploproficient genes showed any enrichment in the budding yeast haploproficient gene set. If fission yeast evolved in a nutrition-poor niche, then these pathways may have evolved to fine-tune optimal growth in these conditions, which may result in a sub-maximal growth rate in rich media. Haploinsufficient genes from budding yeast showed a significant GO enrichment for ribosomal-related function29, whereas those from fission yeast did not (Supplementary Table 18). We reasoned that any genes common to both haploinsufficient gene sets are likely to be important for regulating growth in both yeasts. A comparison of these gene sets in the two yeasts (138 versus 184) revealed 14 common orthologous groups and 15 genes (Table 1b). These included three genes encoding small subunit ribosomal proteins (S3, S6 and S7), five genes encoding large subunit ribosomal proteins (L6, redundant L13, L35 and L39) and another five genes involved in transcriptional functions including a predicted transcription factor TFIID complex subunit A/SAGA complex subunit (taf12), DNA-directed RNA polymerase II–specific subunits, (rpb3 and rpb7) and DNA-directed RNA polymerase subunits (rpb6 and rpc10), which are common to DNA-directed RNA polymerases I, II and III. Because the haploinsufficiency of these genes has been conserved between two distantly related organisms, it is likely that the amount of protein encoded by them is particularly important for the growth rate of the cell. It is therefore possible that their dosage is also important for the regulation of growth in other eukaryotes.
Table 1 haploinsufficient and haploproficient genes in the two yeasts
(a) GO term Translation & ribosome 60S biogenesis (GO:0006412) Haploproficient (HP) gene rpl301, rpl501, rpl702, rpl801, rpl901, rpl902, rpl1001, rpl1101, rpl1701, rpl1801, rpl2001, rpl2002, rpl1901, rpl2101, rpl2102, rpl2301, rpl2502, rpl2802, rpl3001, rpl3201, rpl3202, rpl3401, rpl3601, rpl3602, rpl3702, rpl4301, rpl3801, rpp201 (28 genes) rps001, rps002, rps401, rps402, rps403, rps502, rps801, rps802, rps901, rps1001, rps1002, rps1101, rps1102, rps1201, rps13, rps1501, rps1502, rps1602, rps1701, rps1702, rps1801, rps1902, rps23, rps2302, rps2402, rps2802 (26 genes) gyp1, gyp7, gyp51, SPAC1952.17c tor2, tsc1, mip1, tco89, gad8 HP gene annotation 54 Total gene annotation 316 P-value (uncorrected) 2.10 × 10?19
Regulation of Rab GTPase activity (GO:0032313) TOR signaling pathway (GO:0031929)
5.30 × 10?4 8.55 × 10?3
? 2010 Nature America, Inc. All rights reserved.
Of the genes deleted in the 136 haploproficient mutants with the fastest growth rates, 54 genes (39.7%) encode ribosomal subunit proteins and nine genes encode Rab GAP and TOR pathway-related proteins. For GO enrichment of the haploproficient genes in fission yeast, see Supplementary Tables 17 and 18.
(b) Gene product Ribosomal protein S3 Ribosomal protein S6 Ribosomal protein S7 Ribosomal protein L6 Ribosomal protein L13 Ribosomal protein L35 Ribosomal protein L39 TFIID subunit A (Taf12) RNA pol II Rpb3 RNA pol Rpb6 RNA pol II Rpb7 RNA pol Rpc10 U3 snoRNP Utp4 ATPase Rvb2 Gene category Ribosomal subunit Ribosomal subunit Ribosomal subunit Ribosomal subunit Ribosomal subunit Ribosomal subunit Ribosomal subunit Transcription Transcription Transcription Transcription Transcription RNA processing Chromatin remodeling Budding yeast ID YNL178W YPL090C YNL096C|YOR096W YLR448W|YML073C YDL082W|YMR142C YDL136W|YDL191W YJL189W YDR145W YIL021W YPR187W YDR404C YHR143W-A YDR324C YPL235W Fission yeast ID SPBC16G5.14c SPAPB1E7.12 SPAC18G6.14c SPCC622.18 SPAC664.05|SPBC839.13c SPCC613.05c SPCC663.04 SPAC15A10.02 SPCC1442.10c SPCC1020.04c SPACUNK4.06c SPBC19C2.03 SPBC19F5.02c SPBC83.08 Sc:Sp 1:1 1:1 2:1 2:1 2:2 2:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1
Genes common to the haploinsufficient gene sets of both fission yeast and budding yeast (Supplementary Tables 17 and 18). Of these 15 genes, 13 (86.7%) are involved in transcription or translation.
DISCUSSION Fission yeast is an important model eukaryotic organism and the availability of a genome-wide deletion collection will facilitate further studies such as genetic interaction assays, phenotypic analysis4, comparative genomics, gene dispensability analysis of higher eukaryotes and drug-induced haploinsufficiency screening34. For example, a partial collection of the viable haploid deletions has been distributed to ~25 laboratories and studies from two laboratories have shown that there is considerable conservation of synthetic lethal genetic interactions with budding yeast as well as rewiring of some functionally conserved modules35,36. Our comparisons of orthologous gene pairs between budding and fission yeast showed that 83% had the same dispensability despite being distantly related. This high level of conservation in dispensability will be helpful for the interpretation of more complex RNAi data from other organisms 6,37–39. We have also shown that there is a relationship between gene essentiality and the presence of introns, which may indicate that essential genes are less likely to be rapidly regulated 16. There are orthologs for 3,492 fission yeast genes in other eukaryotes, including humans. Of these genes, 454 are not conserved in budding yeast suggesting that fission yeast may be a valuable alternative organism to budding yeast for certain experiments, for example, optimization of drug screening protocols. However, there are ~3,038 genes conserved in
both yeasts and other eukaryotes including humans, which encourages us in the view that conclusions drawn from analyses in the two yeasts concerning molecular and cell biology will be relevant to, and improve our understanding of, metazoan cells. We have also identified a small set of genes required for translation and transcription, including genes encoding specific ribosomal proteins and RNA polymerase subunits that are haploinsufficient for growth in both the yeasts. These specific gene products may play a critical role in regulating the growth of eukaryotic cells. The identification of genes encoding elements of the TOR pathway, Rab-GTPase activating proteins and ribosomal proteins, as haploproficient, is also of interest given the involvement of these gene products in cancer31–33. The availability of a near-complete, genome-wide deletion collection for fission yeast provides a useful tool for the functional studies of eukaryotic molecular and cell biology and for biotechnological applications. METhODS Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturebiotechnology/.
Note: Supplementary information is available on the Nature Biotechnology website. ACKNOWLEDGMENTS We thank members of our laboratories for their participation in the construction and analysis of the deletion mutants, particularly H.-R. Hwang, H.-S. Ahn,
Y.-D. Kim, S. Park, H.-J. Lee, J.-H. Ahn, Y.-S. Kil, S.-Y. Park, J.-H. Lim, J.-H. Song, Y.-K. Ryoo, J.-Y. Kim, M.-J. Oh, S. Kong, J. Ahn, N. Sun, N. Peat, R. Mandeville and J.-J. Li. We also thank J.-H. Roe and W.-K. Huh for reading this manuscript and for their insightful comments and O. Nielsen for his patience with the many requests for pON177. This work was supported by the intramural research program of KRIBB (Mission 2007), the Chemical Genomics Research Program and the 21st Century Frontier Research Program from the Ministry of Education, Science and Technology (MOEST) of Korea. This work was also supported by Bioneer Corp., The Wellcome Trust, Cancer Research UK, The Breast Cancer Research Foundation (BCRF) and The Rockefeller University. AUTHOR CONTRIBUTIONS D.-U.K., J.H., H.-O.P., M.W., H.-S.Y., P.N. and K.-L.H. conceived the project; D.-U.K., J.H., D.K., V.W., M.W., T.D., M.N., G.P., S.H., L.J., S.-T.B., H.L., Y.S.S., M.L., L.K., K.-S.H., E.J.N., A.-R.L., Y.-J.J., K.-S.C., S.-J.C., J.-Y.P., Y.P., H.M.K., S.-K.P., H.B.K., H.-S.K., H.-M.P., K.K., K.S. and K.B.S. performed experiments and data analysis; D.K., H.-J.P., E.-J.K. and H.-M.P. performed primer design; D.K. and V.W. performed bioinformatics; D.-U.K., J.H., D.K., V.W., P.N. and K.-L.H. wrote the paper. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
14. Sipiczki, M. Where does fission yeast sit on the tree of life? Genome Biol. 1, reviews 1011.1–1011.4 (2000). 15. Wood, V. Schizosaccharomyces pombe comparative genomics; from sequence to systems, in Comparative Genomics: Using Fungi as Models (eds. Sunnerhagen, P. & Piskur, J.), 233–285 (Springer Berlin, Heidelberg, 2006). 16. Jeffares, D.C., Penkett, C.J. & Bahler, J. Rapidly regulated genes are intron poor. Trends Genet. 24, 375–378 (2008). 17. Matsuyama, A. et al. ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat. Biotechnol. 24, 841–847 (2006). 18. Huh, W.K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003). 19. Benton, M.J. & Ayala, F.J. Dating the tree of life. Science 300, 1698–1700 (2003). 20. Hoskin, C.J., Higgie, M., McDonald, K.R. & Moritz, C. Reinforcement drives rapid allopatric speciation. Nature 437, 1353–1356 (2005). 21. Harrison, R., Papp, B., Pal, C., Oliver, S.G. & Delneri, D. Plasticity of genetic interactions in metabolic networks of yeast. Proc. Natl. Acad. Sci. USA 104, 2307–2312 (2007). 22. Chiron, S., Suleau, A. & Bonnefoy, N. Mitochondrial translation: elongation factor tu is essential in fission yeast and depends on an exchange factor conserved in humans but not in budding yeast. Genetics 169, 1891–1901 (2005). 23. Choi, D.H., Oh, Y.M., Kwon, S.H. & Bae, S.H. The mutation of a novel Saccharomyces cerevisiae SRL4 gene rescues the lethality of rad53 and lcd1 mutations by modulating dNTP levels. J. Microbiol. 46, 75–80 (2008). 24. Ralph, E., Boye, E. & Kearsey, S.E. DNA damage induces Cdt1 proteolysis in fission yeast through a pathway dependent on Cdt2 and Ddb1. EMBO Rep. 7, 1134–1139 (2006). 25. Liu, C. et al. Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms. Genes Dev. 17, 1130–1140 (2003). 26. Preuss, D., Mulholland, J., Franzusoff, A., Segev, N. & Botstein, D. Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy. Mol. Biol. Cell 3, 789–803 (1992). 27. Ayscough, K., Hajibagheri, N.M., Watson, R. & Warren, G. Stacking of Golgi cisternae in Schizosaccharomyces pombe requires intact microtubules. J. Cell Sci. 106, 1227–1237 (1993). 28. Roemer, T. et al. Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery. Mol. Microbiol. 50, 167–181 (2003). 29. Deutschbauer, A.M. et al. Mechanisms of haploinsufficiency revealed by genomewide profiling in yeast. Genetics 169, 1915–1925 (2005). 30. Pierce, S.E. et al. A unique and universal molecular barcode array. Nat. Methods 3, 601–603 (2006). 31. Jozwiak, J., Jozwiak, S. & Wlodarski, P. Possible mechanisms of disease development in tuberous sclerosis. Lancet Oncol. 9, 73–79 (2008). 32. Cheng, K.W., Lahad, J.P., Gray, J.W. & Mills, G.B. Emerging role of RAB GTPases in cancer and human disease. Cancer Res. 65, 2516–2519 (2005). 33. McGowan, K.A. et al. Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects. Nat. Genet. 40, 963–970 (2008). 34. Lum, P.Y. et al. Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes. Cell 116, 121–137 (2004). 35. Roguev, A. et al. Conservation and rewiring of functional modules revealed by an epistasis map in fission yeast. Science 322, 405–410 (2008). 36. Dixon, S.J. et al. Significant conservation of synthetic lethal genetic interaction networks between distantly related eukaryotes. Proc. Natl. Acad. Sci. USA 105, 16653–16658 (2008). 37. Kamath, R.S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003). 38. Dietzl, G. et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151–156 (2007). 39. Ravi, D. et al. A network of conserved damage survival pathways revealed by a genomic RNAi screen. PLoS Genet. 5, e1000527 (2009).
? 2010 Nature America, Inc. All rights reserved.
Published online at http://www.nature.com/naturebiotechnology/. reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/.
1. Jorgensen, P. et al. High-resolution genetic mapping with ordered arrays of Saccharomyces cerevisiae deletion mutants. Genetics 162, 1091–1099 (2002). 2. Hillenmeyer, M.E. et al. The chemical genomic portrait of yeast: uncovering a phenotype for all genes. Science 320, 362–365 (2008). 3. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002). 4. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999). 5. Entian, K.D. & Kotter, P. Methods in Microbiology 36, edn. II. 629–666 (Elsevier, 2007). 6. Kittler, R. et al. Genome-scale RNAi profiling of cell division in human tissue culture cells. Nat. Cell Biol. 9, 1401–1412 (2007). 7. Wood, V. et al. The genome sequence of Schizosaccharomyces pombe. Nature 415, 871–880 (2002). 8. Fisk, D.G. et al. Saccharomyces cerevisiae S288C genome annotation: a working hypothesis. Yeast 23, 857–865 (2006). 9. Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808 (1994). 10. Gregan, J. et al. Novel genes required for meiotic chromosome segregation are identified by a high-throughput knockout screen in fission yeast. Curr. Biol. 15, 1663–1669 (2005). 11. Martin-Castellanos, C. et al. A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events. Curr. Biol. 15, 2056–2062 (2005). 12. Decottignies, A., Sanchez-Perez, I. & Nurse, P. Schizosaccharomyces pombe essential genes: a pilot study. Genome Res. 13, 399–406 (2003). 13. Smith, H.O., Hutchison, C.A. III, Pfannkoch, C. & Venter, J.C. Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc. Natl. Acad. Sci. USA 100, 15440–15445 (2003).
nature biotechnology? VOLUME 28
? 2010 Nature America, Inc. All rights reserved.
Construction of genome-wide deletion mutants. Heterozygous deletion mutants of 4,836 protein coding genes in fission yeast were constructed using a method based on homologous recombination of a deletion cassette containing a pair of unique molecular bar codes (up-tag and down-tag in Supplementary Table 1) and the KanMX marker gene9. The sequences of bar codes was generated using a BioPerl-based computer program to meet the following criteria; melting temperature (Tm) = 60 °C, no cross-hybridization, no secondary structures and no similarities to genomic sequences. RNAfold and mfold freeware (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) was used for checking secondary structure, and the BLAST program was used for checking similarity with genomic sequence. Deletion cassettes were generated by a modified PCR-based strategy. For one-third of deletion cassettes, the conventional serial-extension PCR method3,4 was used. For the remaining two-thirds, the block PCR method or an innovative gene synthesis method13 was employed, resulting in the increase in the length of homologous recombination regions from ~80 bp to 250~450 bp. Oligonucleotides used in construction of the deletion cassettes were supplied by Bioneer Corporation. The deletion cassettes were transformed into SP286 (ade6-M210/ade6-M216, leu1-32/leu1-32, ura4-D18/ura4-D18 h+/h+) using a lithium acetate method40, and then incubated for 5 d to select positive colonies on YES agar containing 100 μg/ml G418 (Duchefa Biochemie). Confirmation of genome-wide deletion mutants. To verify the integration of deletion cassettes at the correct locus, colony PCR was carried out. Dideoxy sequencing of the PCR product from each successful deletion mutant was carried out to confirm the sequences of up- and down-tags as well as the junctions to accurately define the deleted region. To estimate how often the deletion cassette integrated at additional sites in the genome, Southern blot analysis of chromosomal DNA from 61 different deletion strains was carried out using KanMX4 as a probe. All the strains and check-PCR primers described here are available from Bioneer (http://pombe.bioneer.co.kr). Determination of essentiality. General growth conditions and media were used as described41. Essentiality was determined by a microscopic observation of colony-forming ability of spores on YES (yeast extract medium supplemented with adenine, leucine, uracil and histidine at 250 mg/l) at 25 °C and 32 °C. The spores were derived from corresponding heterozygous diploid deletion strains transformed with the pON177 plasmid 42 using a modified version of the PLATE method 43. About 5% of the heterozygous deletion diploids could not be transformed using this high-throughput method and these were repeated using a standard transformation protocol40. Briefly, four batches each of 48 heterozygous diploid strains were patched on to YE (yeast extract medium supplemented with leucine and uracil at 250 mg/l) + G418 agar plates in two 96-well microtiter plates (each strain is represented four times) and left to grow for 2~3 d at 32 °C. Cells were inoculated into 200 μl YE + G418 and left to grow into stationary phase. The cells were harvested and transformed with pON177 (ref. 42), plated on minimal agar + leucine (250 mg/l) and incubated for a week at 32 °C. Transformants were inoculated into minimal media lacking nitrogen and left for 2~3 d at 25 °C to induce sporulation. The asci were treated with helicase (Bio Sepra) diluted 1 in 250 to eliminate vegetative cells, washed with water and the haploid spores were plated on YES agar at 25 °C and 32 °C. Essentiality was determined by a microscopic observation of the germinating spores on plates after 1 and 2 d before replica plating to YES +100 μg/ml G418 to confirm that the deletion phenotype was associated with G418 resistance. Essential genes were further analyzed by tetrad analysis. Briefly, cells harboring pON177 were left to germinate for 4~5 d on minimal plates. Using a Singer MSM microscope, spores were dissected on YES plates for 4~5 d at 30 °C. Viable colonies were patched onto YES plates + 100 μg/ml G418 to confirm that viability was linked to G418 sensitivity (Supplementary Methods). While analyzing gene dispensability, we found that a subset of the deletion collection harbored a recessive temperature-sensitive mutation unrelated to the gene deletion. This ts mutation was removed from the entire nonessential haploid deletion library after sporulation of the diploid heterozygous deletion strains of nonessential genes. There were originally 416 of the 1,260 essential heterozygous deletion diploid strains that harboured the ts mutation. Of these 416 strains, 364 have been remade and the remaining 52 are
currently being remade (Supplementary Table 1, column U for the list of heterozygous diploid strains that still contain the ts mutation). Redundancy and essentiality. To assess the effect of redundancy on masking essentiality and its contribution to the extra essential genes in fission yeast, we identified all genes in the one|many, many|one and many|many categories where data were available for both organisms (Supplementary Table 1). We eliminated all orthologous groups with an equal number of essential genes in each organism (e.g., ev|ev) and those where redundancy could not contribute to the difference in essentiality (e.g., vv|vv). The remaining essential genes where redundancy could mask essentiality in one or the other organism were counted for both yeasts. There were 67 essential genes in fission yeast and 35 essential genes in budding yeast where redundancy in the other yeast could potentially be masking essentiality. Data source and URLs. DNA and protein information of fission yeast were from the S. pombe GeneDB database ftp://ftp.sanger.ac.uk/pub/yeast/pombe/ Mappings/OLD/allNames.txt_27Aug2008, and the budding yeast data set from http://www.yeastgenome.org/. Budding yeast deletion data3 were from http://downloads.yeastgenome.org/literature_curation/archive/phenotypes. tab.20080202.gz. Interspecies comparisons used manually curated species distribution from GeneDB on 24/06/2008 and Version 13 of the manually curated fission yeast/budding yeast ortholog table. Distant ortholog detection. The detection of distant orthologs used all essential S. pombe and all S. cerevisiae proteins that were not already members of an existing orthologous group, based on the manually curated S. cerevisae/ S. pombe ortholog table version 13 (ref. 15). Ortholog candidate detection used PSI blast, and the criteria as described15 were used to support orthologous cluster predictions. Individual multiple alignments are provided in Supplementary Table 10. One ortholog prediction SPAC1006.42/YPR085C pair has since been confirmed experimentally (PMID 19040720). GO analysis. GO enrichment analysis used the Princeton implementation of GO term finder44 (http://go.princeton.edu/cgi-bin/GOTermFinder) with gene association files from November 2008; GO TermFinder calculates P-value using the hypergeometric distribution, and Bonferroni method is used for multiple hypothesis correction. Analysis used a P-value cut off of 0.01 and all evidence codes except RCA (reviewed computational analysis) are included. The whole genome comparison in Figure 2d and Supplementary Tables 5 and 6 used the total protein coding data sets for fission yeast (4,836) and budding yeast (5,776). Some biologically uninformative terms were omitted from the results (that is, when parent and child terms show identical enrichment only the child term is included). GO process enrichment of essential genes, which are conserved in single copy (Supplementary Table 12), versus nonessential genes conserved in single copy (Supplementary Table 13) used fission yeast annotations and background set. Parallel analysis using microarray. The custom-made GeneChip (48 K) was designed and manufactured according to the Affymetrix GeneChip guide (KRIBBSP2, Part No. 520506). Construction of mutant library pools, sampling, PCR amplification of probes, hybridization and washes were carried out following modified budding yeast protocols29,30. Genomic DNA was prepared from frozen cell stocks using a kit (Zymo Research ZR-Fungal/Bacterial DNA kit). For each sample, 10~20 OD600 corresponding to 2~4 × 108 cells/ml was used for the genomic DNA preparation. To amplify and label the tags the following sets of primers were used for PCR using 0.2 μg genomic DNA as a template; uptag, forward (5′U-2) 5′-GCTCCCGCCTTACTTCGCAT-3′, reverse (biotin-Kan5′U-2) 5′-biotin-CGGGGACGAGGCAAGCTAA-3′; downtag, forward (DN3-F-biotin) 5′-biotin-GCCGCCATCCAGTGTCG-3′, reverse (DN3-R) 5′-TTGCGTTGCGTAGGGGGG-3′. For growth profiling, data were collected from six independent experiments using two different pool sets. For details, see Supplementary Figures 7–9. Analysis of microarray results. Out of 4,441 mutants in the deletion pool, 3,523 mutants were represented by both up-tag and down-tag, and 811 mutants were represented by at least one of two tags. Therefore, at least one of the tags
from 4,334 strains was detectable by chip analysis. The remaining 107 tags were removed from the analysis, as they had intensities less than fourfold that of background. For the analysis of microarray results, the analysis of covariance (ANCOVA) model was used as a statistical tool. Each array signal was normalized by a mean-intensity (that is, 2,500 arbitrary units) and interpreted by ANCOVA as a linear regression corresponding to a multiple-regression model on time (measured in generations and treated as a quantitative predictor) and replicate series (treated as a categorical predictor) simultaneously. This analysis provides estimates of statistical significance (P-values) using the F-statistic (Supplementary Methods).
40. Bahler, J. et al. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951 (1998). 41. Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991). 42. Styrkarsdottir, U., Egel, R. & Nielsen, O. The smt-0 mutation which abolishes mating-type switching in fission yeast is a deletion. Curr. Genet. 23, 184–186 (1993). 43. Elble, R. A simple and efficient procedure for transformation of yeasts. Biotechniques 13, 18–20 (1992). 44. Boyle, E.I. et al. GO:TermFinder–open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Bioinformatics 20, 3710–3715 (2004).
? 2010 Nature America, Inc. All rights reserved.