Yeast Functional Analysis Report

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Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

  1. Jürg Bähler1,†,§,
  2. Jian-Qiu Wu1,¶,
  3. Mark S. Longtine1,
  4. Nirav G. Shah1,
  5. Amos Mckenzie III1,
  6. Alexander B. Steever1,
  7. Achim Wach1,‡,
  8. Peter Philippsen2,
  9. John R. Pringle1,*

Article first published online: 4 DEC 1998

DOI: 10.1002/(SICI)1097-0061(199807)14:10<943::AID-YEA292>3.0.CO;2-Y

Issue

Yeast

Yeast

Volume 14, Issue 10, pages 943–951, July 1998

Additional Information

How to Cite

Bähler, J., Wu, J.-Q., Longtine, M. S., Shah, N. G., Mckenzie III, A., Steever, A. B., Wach, A., Philippsen, P. and Pringle, J. R. (1998), Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast, 14: 943–951. doi: 10.1002/(SICI)1097-0061(199807)14:10<943::AID-YEA292>3.0.CO;2-Y

Author Information

  1. 1

    Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.

  2. 2

    Institut für Angewandte Mikrobiologie, Biozentrum, Universität Basel, CH-4056 Basel, Switzerland

  1. †

    Imperial Cancer Research Fund, Cell Cycle Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.

  2. ‡

    Bureco AG, Stadtweg 4, CH-4310 Rheinfelden, Switzerland

  1. §

    Jürg Bähler and Jian-Qiu Wu contributed equally to this work.

  2. Jürg Bähler and Jian-Qiu Wu contributed equally to this work.

*Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.

Publication History

  1. Issue published online: 4 DEC 1998
  2. Article first published online: 4 DEC 1998
  3. Manuscript Accepted: 25 FEB 1998
  4. Manuscript Received: 24 DEC 1997

Funded by

  • NIH. Grant Number: GM31006
  • RJEG Trust
  • University of Basel
  • Swiss Federal Office for Education and Science. Grant Number: 95.0191

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Keywords:

  • fission yeast;
  • gene deletions;
  • gene truncations;
  • overexpression studies;
  • epitope tagging;
  • polymerase chain reaction;
  • gene expression;
  • green fluorescent protein

Abstract

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe. © 1998 John Wiley & Sons, Ltd.

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