Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe

Journal name:
Nature Biotechnology
Volume:
28,
Pages:
617–623
Year published:
DOI:
doi:10.1038/nbt.1628
Received
Accepted
Published online
Corrected online

Abstract

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.

At a glance

Figures

  1. Deletion construction and gene dispensability.
    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.

  2. Analysis of gene dispensability.
    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.

  3. Comparative analysis of gene dispensability profiles of fission yeast.
    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.

  4. Dispensability comparison of orthologous pairs from the two yeasts.
    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.

  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).
    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).

Change history

Corrected online 07 December 2010
In the version of this article initially published, the address of one of the authors, Young-Joo Jang, was incorrect. The correct address is Laboratory of Cell Cycle & Signal Transduction, WCU Department of NanoBioMedical Science, Institute of Tissue Regeneration Engineering, Dankook University, Cheonan, Korea.The error has been corrected in the HTML and PDF versions of the article.

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Author information

  1. These authors contributed equally to this work.

    • Dong-Uk Kim,
    • Jacqueline Hayles,
    • Dongsup Kim,
    • Valerie Wood,
    • Han-Oh Park,
    • Misun Won &
    • Hyang-Sook Yoo

Affiliations

  1. Integrative Omics Research Centre, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong, Daejeon, Korea.

    • Dong-Uk Kim,
    • Misun Won,
    • Hyang-Sook Yoo,
    • Miyoung Nam,
    • Seung-Tae Baek,
    • Hyemi Lee,
    • Young Sam Shim,
    • Lila Kim,
    • Kyung-Sun Heo,
    • Eun Joo Noh,
    • Ah-Reum Lee,
    • Kyung-Sook Chung,
    • Shin-Jung Choi,
    • Jo-Young Park,
    • Youngwoo Park &
    • Kwang-Lae Hoe
  2. Cancer Research UK, The London Research Institute, London, UK.

    • Jacqueline Hayles,
    • Valerie Wood,
    • Trevor Duhig,
    • Georgia Palmer,
    • Linda Jeffery &
    • Paul Nurse
  3. Department of Bio and Brain Engineering, Korea Advanced Institute of Science & Technology (KAIST), Yuseong, Daejeon, Korea.

    • Dongsup Kim,
    • Sangjo Han &
    • Minho Lee
  4. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

    • Valerie Wood
  5. Bioneer Corp., Daedeok, Daejeon, Korea.

    • Han-Oh Park,
    • Hae-Joon Park &
    • Eun-Jung Kang
  6. Bioevaluation Centre, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Chungcheongbuk-do, Korea.

    • Hwan Mook Kim,
    • Song-Kyu Park &
    • Kwang-Lae Hoe
  7. Department of Bioinformatics & Biotechnology, Korea University, Jochiwon, Chungnam, Korea.

    • Hyong Bai Kim
  8. School of Biological Sciences, Seoul National University, Seoul, Korea.

    • Hyun-Sam Kang
  9. Department of Microbiology, Chungnam National University, Yuseong, Daejeon, Korea.

    • Hee-Moon Park
  10. Division of Life Sciences, Kangwon National University, Chuncheon, Kangwon-do, Korea.

    • Kyunghoon Kim
  11. Department of Biochemistry, Yonsei University, Seoul, Korea.

    • Kiwon Song
  12. Department of Food and Nutrition, Chungnam National University, Yuseong, Daejeon, Korea.

    • Kyung Bin Song
  13. The Rockefeller University, New York, New York, USA.

    • Paul Nurse
  14. Laboratory of Cell Cycle & Signal Transduction, WCU Department of NanoBioMedical Science, Institute of Tissue Regeneration Engineering, Dankook University, Cheonan, Korea.

    • Young-Joo Jang

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.

Corresponding author

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Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (5M)

    Supplementary Tables 2–10,12–14,18 Supplementary Figs. 1–10 and Supplementary Methods

  2. Supplementary Data 2 (19M)

    Mapping of the deletions

  3. Supplementary Data 3 (232K)

    Sequence of KanMX4

Excel files

  1. Supplementary Table 1 (2M)

    The 4,836 deletion set in fission yeast and its genome dataset as a reference (4,914)

  2. Supplementary Table 11 (576K)

    Spreadsheet of 2,438 'one to one' orthologous pairs in fission yeast and budding yeast (for details, see attached Excel file)

  3. Supplementary Table 15 (1M)

    Growth fitness data of S. pombe heterozygous deletion mutants in rich YE media

  4. Supplementary Table 16 (264K)

    List of slow growers from the two yeasts, whose relative fitness is less than 0.97

  5. Supplementary Table 17 (220K)

    List of haploinsufficient (lowest 3% ranked by RF) and haploproficient (highest 3% ranked by RF) genes from the two yeasts in rich media

  6. Supplementary Data 1 (3M)

    All the primer set for the construction of deletion strains

  7. Supplementary Data 4 (2M)

    Design of Affymertix custom GeneChip

Zip files

  1. Supplementary Data 5 (10M)

    Microarray data set for growth profiling

Additional data