Skip to main content
SpringerLink
Log in

Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe

  • Technical Notes
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

Gene targeting provides a powerful tool to modify endogenous loci to contain specific mutations, insertions and deletions. Precise allele replacement, with no other chromosomal changes (e.g., insertion of selectable markers or heterologous promoters), maintains physiologically relevant context. Established methods for precise allele replacement in fission yeast employ two successive rounds of transformation and homologous recombination and require genotyping at each step. The relative efficiency of homologous recombination is low and a high rate of false positives during the second round of gene targeting further complicates matters. We report that pop-in, pop-out allele replacement circumvents these problems. We present data for 39 different allele replacements, involving simple and complex modifications at seven different target loci, that illustrate the power and utility of the approach. We also developed and validated a rapid, efficient process for precise allele replacement that requires only one round each of transformation and genotyping. We show that this process can be applied in population scale to an individual target locus, without genotyping, to identify clones with an altered phenotype (targeted forward genetics). It is therefore suitable for saturating, in situ, locus-specific mutation screens (e.g., of essential or non-essential genes and regulatory DNA elements) within normal chromosomal context.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Notes

  1. These three comparisons are based on a single allele replacement at the target locus. If multiple alleles are targeted to the same locus, and if the same ura4 + transgene can be used for each allele replacement in the two-step method, then the relative effort (E) of the pop-in, pop-out approach to the two-step approach is approximated by the equation [E = (n)/(n + 1)], where n is number of allele replacements.

References

  • Ahmed S, Brickner DG, Light WH, Cajigas I, McDonough M, Froyshteter AB, Volpe T, Brickner JH (2010) DNA zip codes control an ancient mechanism for gene targeting to the nuclear periphery. Nat Cell Biol 12:111–118

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Bach ML (1987) Cloning and expression of the OMP decarboxylase gene URA4 from Schizosaccharomyces pombe. Curr Genet 12:527–534

    Article  PubMed  CAS  Google Scholar 

  • Bähler J, Wu J-Q, Longtine MS, Shah NG, McKenzie A III, Steever AB, Wach A, Philippsen P, Pringle JR (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14:943–951

    Article  PubMed  Google Scholar 

  • Barton MC, Hoekstra MF, Emerson BM (1990) Site-directed, recombination-mediated mutagenesis of a complex gene locus. Nucleic Acids Res 18:7349–7355

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Bryksin AV, Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 48:463–465

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Davidson MK, Young NP, Glick GG, Wahls WP (2004) Meiotic chromosome segregation mutants identified by insertional mutagenesis of fission yeast Schizosaccharomyces pombe; tandem-repeat, single-site integrations. Nucleic Acids Res 32:4400–4410

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Forsburg SL, Rhind N (2006) Basic methods for fission yeast. Yeast 23:173–183

    Article  PubMed  CAS  Google Scholar 

  • Gao J, Davidson MK, Wahls WP (2008) Distinct regions of ATF/CREB proteins Atf1 and Pcr1 control recombination hotspot ade6-M26 and the osmotic stress response. Nucleic Acids Res 36:2838–2851

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Geiser M, Cebe R, Drewello D, Schmitz R (2001) Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase. Biotechniques 31(88–90):92

    Google Scholar 

  • Grallert B, Nurse P, Patterson TE (1993) A study of integrative transformation in Schizosaccharomyces pombe. Mol Gen Genet 238:26–32

    PubMed  CAS  Google Scholar 

  • Grimm C, Kohli J (1988) Observations on integrative transformation in Schizosaccharomyces pombe. Mol Gen Genet 215:87–93

    Article  PubMed  CAS  Google Scholar 

  • Grimm C, Kohli J, Murray J, Maundrell K (1988) Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet 215:81–86

    Article  PubMed  CAS  Google Scholar 

  • Gutz H, Heslot H, Leupold U, Loprieno N (1974) Schizosaccharomyces pombe. In: King RC (ed) Handbook of genetics. Plenum Press, New York, pp 395–446

    Google Scholar 

  • Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2:924–932

    Article  PubMed  CAS  Google Scholar 

  • Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    PubMed Central  PubMed  CAS  Google Scholar 

  • Ivey FD, Taglia FX, Yang F, Lander MM, Kelly DA, Hoffman CS (2010) Activated alleles of the Schizosaccharomyces pombe gpa2 + Galpha gene identify residues involved in GDP-GTP exchange. Eukaryot Cell 9:626–633

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Kan F, Davidson MK, Wahls WP (2011) Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision. Nucleic Acids Res 39:1460–1472

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Kiely J, Haase SB, Russell P, Leatherwood J (2000) Functions of fission yeast orp2 in DNA replication and checkpoint control. Genetics 154:599–607

    PubMed Central  PubMed  CAS  Google Scholar 

  • Krawchuk MD, Wahls WP (1999) High-efficiency gene targeting in Schizosaccharomyces pombe using a modular, PCR-based approach with long tracts of flanking homology. Yeast 14:1419–1427

    Article  Google Scholar 

  • Mudge DK, Hofman CA, Lubinski TJ, Hofman CS (2012) Use of a ura5 +-lys7 + cassette to construct unmarked gene knock-ins in Schizosaccharomyces pombe. Curr Genet 58:59–64

    Article  PubMed  CAS  Google Scholar 

  • Paluh JL, Clayton DA (1996) Mutational analysis of the gene for Schizosaccharomyces pombe RNase MRP RNA, mrp1, using plasmid shuffle by counterselection on canavanine. Yeast 12:1393–1405

    Article  PubMed  CAS  Google Scholar 

  • Sabatinos SA, Forsburg SL (2010) Molecular genetics of Schizosaccharomyces pombe. Methods Enzymol 470:759–795

    Article  PubMed  CAS  Google Scholar 

  • Schuchert P, Langsford M, Kaslin E, Kohli J (1991) A specific DNA sequence is required for high frequency of recombination in the ade6 gene of fission yeast. EMBO J 10:2157–2163

    PubMed Central  PubMed  CAS  Google Scholar 

  • Sharif WD, Glick GG, Davidson MK, Wahls WP (2002) Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II. Cell Chromosome 1:1

    Article  PubMed Central  PubMed  Google Scholar 

  • Singh SK, Sabatinos S, Forsburg S, Bastia D (2010) Regulation of replication termination by Reb1 protein-mediated action at a distance. Cell 142:868–878

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Steiner WW, Steiner EM, Girvin AR, Plewik LE (2009) Novel nucleotide sequence motifs that produce hotspots of meiotic recombination in Schizosaccharomyces pombe. Genetics 182:459–469

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Steiner WW, Davidow PA, Bagshaw AT (2011) Important characteristics of sequence-specific recombination hotspots in Schizosaccharomyces pombe. Genetics 187:385–396

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Szankasi P, Heyer WD, Schuchert P, Kohli J (1988) DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe. Wild-type and mutant alleles including the recombination hot spot allele ade6-M26. J Mol Biol 204:917–925

    Article  PubMed  CAS  Google Scholar 

  • Tatebayashi K, Kato J, Ikeda H (1994) Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe. Mol Gen Genet 244:111–119

    Article  PubMed  CAS  Google Scholar 

  • Thomson JG, Ow DW (2006) Site-specific recombination systems for the genetic manipulation of eukaryotic genomes. Genesis 44:465–476

    Article  PubMed  CAS  Google Scholar 

  • Watson AT, Garcia V, Bone N, Carr AM, Armstrong J (2008) Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe. Gene 407:63–74

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Charlie Albright for helpful suggestions and Michelle Krawchuk and Wallace Sharif for assistance with method development. This work was supported by Grants from the National Institutes of Health (GM81766) and the UAMS Graduate Student Research Fund. Core facilities were supported in part by a National Institutes of Health Translational Research Institute grant (TR000039).

Author information

Author notes

  1. Jacy L. Wagnon

    Present address: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME, 04609-1500, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street (slot 516), Little Rock, AR, 72205-7199, USA

    Jun Gao, Fengling Kan, Jacy L. Wagnon, Aaron J. Storey, Reine U. Protacio, Mari K. Davidson & Wayne P. Wahls

Authors
  1. Jun Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fengling Kan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jacy L. Wagnon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aaron J. Storey
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Reine U. Protacio
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mari K. Davidson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wayne P. Wahls
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wayne P. Wahls.

Additional information

Communicated by C. S. Hoffman.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 510 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gao, J., Kan, F., Wagnon, J.L. et al. Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe . Curr Genet 60, 109–119 (2014). https://doi.org/10.1007/s00294-013-0406-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-013-0406-x

Keywords

Search

Navigation