Journal ArticleDOI
Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae.
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TLDR
GFP reporters consist of wild‐type GFP or GFP‐S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator and PCR‐synthesized 2·4 kb‐long double modules flanked by 40 bp‐long guide sequences were successfully targeted to the carboxy‐terminus of a number of S. Cerevisiae genes.Abstract:
We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosac-charomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1.4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0.9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2.4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter.read more
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Journal ArticleDOI
Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae
Mark S. Longtine,Amos Mckenzie,Douglas J. Demarini,Nirav Shah,Achim Wach,Arndt Brachat,Peter Philippsen,John R. Pringle +7 more
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.
Journal ArticleDOI
Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis
Elizabeth A. Winzeler,Daniel D. Shoemaker,Anna Astromoff,Hong Liang,Keith Anderson,Bruno André,Rhonda Bangham,Rocío Benito,Jef D. Boeke,Howard Bussey,Angela M. Chu,Carla Connelly,Karen Davis,Fred S. Dietrich,Sally Dow,Mohamed El Bakkoury,Françoise Foury,Stephen H. Friend,Erik Gentalen,Guri Giaever,Johannes H. Hegemann,Ted Jones,Michael T. Laub,Hong Liao,Nicole Liebundguth,David J. Lockhart,Anca Lucau-Danila,Marc Lussier,Nasiha M'Rabet,Patrice Menard,Michael Mittmann,Chai Pai,Corinne Rebischung,José L. Revuelta,Linda Riles,Christopher J. Roberts,Petra Ross-Macdonald,Bart Scherens,Michael Snyder,Sharon Sookhai-Mahadeo,Reginald Storms,Steeve Veronneau,Marleen Voet,Guido Volckaert,Teresa R. Ward,Robert W. Wysocki,Grace Yen,Kexin Yu,Katja Zimmermann,Peter Philippsen,Mark Johnston,Ronald W. Davis +51 more
TL;DR: A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome), finding that 17 percent were essential for viability in rich medium.
Journal ArticleDOI
Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe
Jürg Bähler,Jian-Qiu Wu,Mark S. Longtine,Nirav Shah,Amos Mckenzie,Alexander B. Steever,Achim Wach,Peter Philippsen,John R. Pringle +8 more
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.
Journal ArticleDOI
A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.
Carsten Janke,Maria M. Magiera,Nicole Rathfelder,Christof Taxis,Simone Reber,Hiromi Maekawa,Alexandra C. Moreno-Borchart,Georg Doenges,Etienne Schwob,Elmar Schiebel,Michael Knop,Michael Knop +11 more
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.
Journal ArticleDOI
A NEW APPROACH TO DECODING LIFE: Systems Biology
TL;DR: The emergence of systems biology is described, as well as several examples of specific systems approaches.
References
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Book
Molecular Cloning: A Laboratory Manual
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Journal ArticleDOI
Green fluorescent protein as a marker for gene expression
TL;DR: A complementary DNA for the Aequorea victoria green fluorescent protein produces a fluorescent product when expressed in prokaryotic or eukaryotic cells, which can be used to monitor gene expression and protein localization in living organisms.
Journal ArticleDOI
New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae
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.
Journal ArticleDOI
Wavelength mutations and posttranslational autoxidation of green fluorescent protein
TL;DR: The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.
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