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Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. ​

Literature Information ​

DOI10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U
PMID9717241
JournalYeast (Chichester, England)
Impact Factor2.6
JCR QuartileQ3
Publication Year1998
Times Cited3492
KeywordsPCR-mediated technique, gene deletion, gene modification, Saccharomyces cerevisiae, modular plasmids
Literature TypeJournal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.
ISSN0749-503X
Pages953-61
Issue14(10)
AuthorsM S Longtine, A McKenzie, D J Demarini, N G Shah, A Wach, A Brachat, P Philippsen, J R Pringle

TL;DR ​

This study presents a novel set of plasmids designed for one-step PCR-mediated gene deletion and modification in Saccharomyces cerevisiae, enabling rapid and versatile gene manipulations without the need for plasmid clones. The modular design of these plasmids, which includes various selectable markers and tagging options, significantly enhances the efficiency of functional analysis of genes, thereby advancing research in yeast genetics.

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PCR-mediated technique · gene deletion · gene modification · Saccharomyces cerevisiae · modular plasmids

Abstract ​

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.

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Primary Questions Addressed ​

  1. What are the specific advantages of using the TRP1 gene as a selectable marker in gene modification?
  2. How do the different epitope tags (GFP, GST, 3HA, 13Myc) affect the functionality and detection of proteins in S. cerevisiae?
  3. In what ways can the modular design of these plasmids improve the efficiency of gene manipulation compared to traditional methods?
  4. What are the potential applications of the one-step PCR-mediated technique in other organisms beyond S. cerevisiae?
  5. How does the use of regulatable promoters like GAL1 enhance the control of gene overexpression in experimental settings?

Key Findings ​

Key Insights ​

  1. Research Background and Objective: The study aims to enhance the functional analysis of genes in Saccharomyces cerevisiae by improving gene manipulation techniques. Traditional methods for gene deletion and modification often rely on plasmid cloning, which can be time-consuming and complex. This research introduces a novel one-step PCR-mediated technique that streamlines the process, allowing for quicker gene manipulations without the need for plasmid clones. The objective is to provide a versatile and economical toolkit for researchers to efficiently conduct a variety of gene modifications in yeast.

  2. Major Methods and Findings: The authors describe a new series of plasmids designed to act as templates for PCR synthesis, facilitating multiple gene alteration strategies. These plasmids incorporate selectable markers such as the S. cerevisiae TRP1 gene, along with modules from Schizosaccharomyces pombe (his5+) and Escherichia coli (kan(r)). The study demonstrates that these plasmids can be employed for various applications, including:

    • Gene deletion and overexpression via the regulatable GAL1 promoter.
    • C- or N-terminal tagging of proteins using fluorescent (GFP) and epitope tags (3HA, 13Myc).
    • Partial deletions of protein coding sequences, with options for concurrent tagging. The modular design of the plasmids allows researchers to utilize a limited number of PCR primers to execute a wide range of gene modifications efficiently.

  3. Core Conclusions: The introduction of these plasmids marks a significant advancement in genetic manipulation of S. cerevisiae. The one-step PCR-mediated approach enables rapid and diverse gene modifications, making it a powerful tool for functional genomics. The ability to perform multiple types of modifications using a streamlined process reduces both time and resource expenditure in genetic research.

  4. Research Significance and Impact: This study provides a critical resource for geneticists and molecular biologists working with S. cerevisiae, a model organism widely used in research. The versatility and economy of the new plasmid system are expected to enhance the pace of gene function analysis, facilitating new discoveries in yeast genetics. Furthermore, the techniques developed may inspire similar advancements in other organisms, potentially broadening the impact of this research within the field of molecular biology. Overall, this work contributes to a more efficient framework for gene editing, which could accelerate innovation in biotechnology and related applications.

Literatures Citing This Work ​

  1. The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization. - B L Anderson;I Boldogh;M Evangelista;C Boone;L A Greene;L A Pon - The Journal of cell biology (1998)
  2. Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis. - E Bi;P Maddox;D J Lew;E D Salmon;J N McMillan;E Yeh;J R Pringle - The Journal of cell biology (1998)
  3. Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function. - M S Longtine;H Fares;J R Pringle - The Journal of cell biology (1998)
  4. Oxidative stress-induced destruction of the yeast C-type cyclin Ume3p requires phosphatidylinositol-specific phospholipase C and the 26S proteasome. - K F Cooper;M J Mallory;R Strich - Molecular and cellular biology (1999)
  5. Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism. - J M Galan;M Peter - Proceedings of the National Academy of Sciences of the United States of America (1999)
  6. Nuclear export of Far1p in response to pheromones requires the export receptor Msn5p/Ste21p. - M Blondel;P M Alepuz;L S Huang;S Shaham;G Ammerer;M Peter - Genes & development (1999)
  7. The morphogenesis checkpoint in Saccharomyces cerevisiae: cell cycle control of Swe1p degradation by Hsl1p and Hsl7p. - J N McMillan;M S Longtine;R A Sia;C L Theesfeld;E S Bardes;J R Pringle;D J Lew - Molecular and cellular biology (1999)
  8. High-efficiency gene targeting in Schizosaccharomyces pombe using a modular, PCR-based approach with long tracts of flanking homology. - M D Krawchuk;W P Wahls - Yeast (Chichester, England) (1999)
  9. Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase. - D Huang;G Patrick;J Moffat;L H Tsai;B Andrews - Proceedings of the National Academy of Sciences of the United States of America (1999)
  10. Essential function of the polo box of Cdc5 in subcellular localization and induction of cytokinetic structures. - S Song;T Z Grenfell;S Garfield;R L Erikson;K S Lee - Molecular and cellular biology (2000)

... (3482 more literatures)

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