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

doi:10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U

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

Journal Article•DOI•

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

Mark S. Longtine¹, Amos Mckenzie¹, Douglas J. Demarini¹, Nirav Shah¹, Achim Wach², Arndt Brachat², Peter Philippsen², John R. Pringle¹ - Show less +4 more•Institutions (2)

University of North Carolina at Chapel Hill¹, University of Basel²

01 Jul 1998-Yeast (Yeast)-Vol. 14, Iss: 10, pp 953-961

TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.

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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 eYcient 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 ? 1998 John Wiley & Sons, Ltd

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Journal Article•DOI•

Global analysis of protein localization in budding yeast

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Won-Ki Huh¹, James V. Falvo¹, Luke C. Gerke¹, Adam S. Carroll¹, Russell W. Howson¹, Jonathan S. Weissman¹, Erin K. O'Shea¹ - Show less +3 more•Institutions (1)

University of California, San Francisco¹

16 Oct 2003-Nature

TL;DR: The construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.

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Abstract: A fundamental goal of cell biology is to define the functions of proteins in the context of compartments that organize them in the cellular environment. Here we describe the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins. We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories, and provide localization information for 70% of previously unlocalized proteins. Analysis of this high-resolution, high-coverage localization data set in the context of transcriptional, genetic, and protein-protein interaction data helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.

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4,310 citations

Cites methods from "Additional modules for versatile an..."

...These primers were used to amplify the GFP tag and an auxotrophic marker from a plasmid templat...
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Journal Article•DOI•

Global analysis of protein expression in yeast

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Sina Ghaemmaghami¹, Won-Ki Huh¹, Kiowa Bower¹, Russell W. Howson¹, Archana Belle¹, Noah Dephoure¹, Erin K. O'Shea¹, Jonathan S. Weissman¹ - Show less +4 more•Institutions (1)

University of California, San Francisco¹

16 Oct 2003-Nature

TL;DR: A Saccharomyces cerevisiae fusion library is created where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location, and it is found that about 80% of the proteome is expressed during normal growth conditions.

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Abstract: The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.

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3,894 citations

Cites background from "Additional modules for versatile an..."

...Each of the oligonucleotide pairs have shared 3′ ends that allow for polymerase chain reaction (PCR) amplification of a common insertion cassette, as well as gene-specific 5′ ends that allow for the precise introduction, through homologous recombination, of the amplified insertion cassettes as a perfect in-frame fusion at the carboxy-terminal end of the coding region of each gen...
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Journal Article•DOI•

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

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Jürg Bähler¹, Jian-Qiu Wu¹, Mark S. Longtine¹, Nirav Shah¹, Amos Mckenzie¹, Alexander B. Steever¹, Achim Wach¹, Peter Philippsen², John R. Pringle¹ - Show less +5 more•Institutions (2)

University of North Carolina at Chapel Hill¹, University of Basel²

01 Jul 1998-Yeast

TL;DR: A straightforward PCR‐based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe, and 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. pom be.

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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 > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

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2,212 citations

Journal Article•DOI•

Functional and Genomic Analyses Reveal an Essential Coordination between the Unfolded Protein Response and ER-Associated Degradation

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Kevin J. Travers¹, Christopher K Patil¹, Lisa Wodicka², David J. Lockhart², Jonathan S. Weissman¹, Peter Walter¹ - Show less +2 more•Institutions (2)

University of California, San Francisco¹, Affymetrix²

28 Apr 2000-Cell

TL;DR: The unfolded protein response and ERAD are dynamic responses required for the coordinated disposal of misfolded proteins even in the absence of acute stress.

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1,941 citations

Cites methods from "Additional modules for versatile an..."

...Time points were taken by transferring 1 ml aliquots intoan auxotrophic marker (LEU2 for PER100 or HIS3 for HRD1 and HRD3) chilled tubes containing 100 ml 100% TCA and 3 ml 3M sodiumusing the method of Pringle and coworkers (Longtine et al., 1998). azide and freezing immediately in liquid nitrogen....
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Journal Article•DOI•

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.

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Carsten Janke¹, Maria M. Magiera¹, Nicole Rathfelder², Christof Taxis², Simone Reber², Hiromi Maekawa, Alexandra C. Moreno-Borchart³, Georg Doenges³, Etienne Schwob¹, Elmar Schiebel, Michael Knop², Michael Knop³ - Show less +8 more•Institutions (3)

Centre national de la recherche scientifique¹, European Bioinformatics Institute², Max Planck Society³

01 Aug 2004-Yeast

TL;DR: Using the provided cassettes for N‐ and C‐terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost‐effective and reproducible.

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Abstract: Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.

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1,931 citations

Cites methods from "Additional modules for versatile an..."

...…has been widely used for research, particularly with the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe (Bahler et al., 1998; Baudin et al., 1993; Knop et al., 1999; Krawchuk and Wahls, 1999; Longtine et al., 1998; Schneider et al., 1995; Tasto et al., 2001; Wach et al., 1994, 1997)....
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...The targeted introduction of heterologous DNA to genomic locations by a simple polymerase chain reaction (PCR)-based strategy has been widely used for research, particularly with the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe (Bahler et al., 1998; Baudin et al., 1993; Knop et al., 1999; Krawchuk and Wahls, 1999; Longtine et al., 1998; Schneider et al., 1995; Tasto et al., 2001; Wach et al., 1994, 1997)....
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References

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Journal Article•DOI•

A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

[...]

Robert S. Sikorski¹, Philip Hieter¹•Institutions (1)

Johns Hopkins University School of Medicine¹

01 May 1989-Genetics

TL;DR: A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae to perform most standard DNA manipulations in the same plasmid that is introduced into yeast.

...read moreread less

Abstract: A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.

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8,364 citations

"Additional modules for versatile an..." refers methods in this paper

...To construct plasmid pFA6a-TRP1, the 2920-bp PCR product obtained using pRS304 (Sikorski and Hieter, 1989) as template, forward primer 5*-AAAAGATCTGTACAATCTTGATC CGGAGC-3*, and reverse primer 5*-AAAGTTT AAACCTCCTTACGCATCTGTGCGG-3* was digested with BglII and PmeI and ligated into BglII/PmeI-digested pFA6a-kanMX6, thus replacing the kanMX6 module with the S....
[...]
...A disadvantage of using the TRP1 marker in this method is that it can recombine efficiently with the endogenous trp1 locus; thus it is only practical for use in strains that contain TRP1 deletions such as trp1Ä-63 (Sikorski and Hieter, 1989)....
[...]
...To construct plasmid pFA6a-TRP1, the 2920-bp PCR product obtained using pRS304 (Sikorski and Hieter, 1989) as template, forward primer 5*-AAAAGATCTGTACAATCTTGATC CGGAGC-3*, and reverse primer 5*-AAAGTTT AAACCTCCTTACGCATCTGTGCGG-3* was digested with BglII and PmeI and ligated into…...
[...]

Journal Article•

A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

[...]

R Sikorski, Philip Hieter, Robert S. Sikorski, Daniel Wai Hung Ho

29 Apr 1989-Genomics

7,587 citations

Journal Article•DOI•

Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.

[...]

D B Smith¹, Kevin S. Johnson²•Institutions (2)

Walter and Eliza Hall Institute of Medical Research¹, University of Melbourne²

15 Jul 1988-Gene

TL;DR: Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum.

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6,003 citations

"Additional modules for versatile an..." refers background or methods in this paper

...1 15X Myc (containing sequences encoding 15 tandem repeats of the Myc epitope) was kindly provided by O. Mondesert and P. Russell; plasmid pGEX-2T (Smith and Johnson, 1988) contains the Yeast 14, 953–961 (1998) ?...
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...…both the localization of proteins by immunofluorescence (Bi and Pringle, 1996; Longtine et al., 1998) and the rapid, one-step biochemical purification of both the tagged protein and associated proteins using glutathione-conjugated agarose beads (Smith and Johnson, 1988; Ausubel et al., 1995)....
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...Yeast 14, 953–961 (1998) Additional Modules for Versatile and Economical PCR-based Gene Deletion and Modification in...
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Journal Article•DOI•

Improved method for high efficiency transformation of intact yeast cells.

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D. Gietz¹, A. Saint Jean², Robin A. Woods³, Robert H. Schiestl³•Institutions (3)

University of Manitoba¹, University of Winnipeg², Harvard University³

25 Mar 1992-Nucleic Acids Research

3,262 citations

"Additional modules for versatile an..." refers methods in this paper

...This concentrated DNA was transformed into S. cerevisiae cells using a lithium acetate procedure (Gietz et al., 1992)....
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...cerevisiae cells using a lithium acetate procedure (Gietz et al., 1992)....
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Journal Article•DOI•

New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae

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Achim Wach¹, Arndt Brachat¹, Rainer Pöhlmann¹, Peter Philippsen¹•Institutions (1)

University of Basel¹

01 Dec 1994-Yeast

TL;DR: A dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA is constructed and tested, and some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10–3–10–4.

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Abstract: We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.

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2,727 citations

"Additional modules for versatile an..." refers background or methods in this paper

...Recently, a PCR-mediated technique has been developed that allows single-step deletion and tagging of chromosomal genes (McElver and Weber, 1992; Baudin et al., 1993; Lorenz et al., 1995; Wach et al., 1994, 1997, 1998)....
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...selectable markers including the kanMX6 module (Wach et al., 1994), the S....
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...PCR template plasmids for gene deletion and gene tagging Wach et al. (1994, 1997) have described a set of plasmids that can be used as templates in PCR reactions to generate DNA fragments that can be used for targeted modification of chromosomal genes....
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...…marker modules based on the Escherichia coli kanr gene (which confers resistance to G418/geneticin: Jiminez and Davies, 1980; Hadfield et al., 1990) or the Schizosaccharomyces pombe his5+ gene (which complements S. cerevisiae his3 mutations) have been developed (Wach et al., 1994, 1997)....
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...G418resistant transformants (containing the kanMX6 module) were selected essentially as described previously (Wach et al., 1994; Wach, 1996)....
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