Exploration of Essential Gene Functions via Titratable Promoter Alleles

Mnaimneh, Sanié; Davierwala, Armaity P.; Haynes, Jennifer; Moffat, Jason; Peng, Wentao; Zhang, Wen; Yang, Xueqi; Pootoolal, Jeff; Chua, Gordon; López, Andrés Aguilera; Trochesset, Miles; Morse, Darcy L; Krogan, Nevan J.; Hiley, Shawna L.; Z, Li; Morris, Quaid; Grigull, Jörg; Mitsakakis, Nicholas; Roberts, Christopher J.; Greenblatt, Jack; Boone, Charles; Kaiser, Chris A.; Andrews, Brenda; Hughes, Timothy R.

doi:10.1016/j.cell.2004.06.013

Cited by 551 publications

(585 citation statements)

References 59 publications

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“…Our vectors can integrate into commonly used auxotrophic yeast strains, including the designer deletion strains (Brachmann et al, 1998), making them compatible with the diverse strain collections (Winzeler et al, 1999;Huh et al, 2003;Ghaemmaghami et al, 2003;Mnaimneh et al, 2004). Efficient integration into the various alleles (carrying point mutations, insertions, partial or complete deletions) is facilitated by using marker genes on the plasmids without any overlap with the host genome (Wach et al, 1997;Goldstein et al, 1999;Gueldener et al, 2002 For the extrachromosomal maintenance of genes at low or high copy number, the series contains centromeric and episomal shuttle vectors, respectively.…”

Section: Discussionmentioning

confidence: 99%

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Gnügge

Liphardt

Rudolf

2016

Yeast

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Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, such as limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series, consisting of single-and multi-copy integrative, centromeric and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double-crossover recombination mechanism, resulting in stable single-and multi-copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behaviour was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with improved copy number control.

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Section: Discussionmentioning

confidence: 99%

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Gnügge

Liphardt

Rudolf

2016

Yeast

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“…With promoter replacement alleles each open reading frame (ORF) remains intact, and repression conditions with minimal effects on cell physiology can be chosen. In a previous study (Mnaimneh et al, 2004), we described the construction of tetracycline-regulatable promoter (TetO 7 promoter) alleles of ϳ600 essential genes in S. cerevisiae. The TetO 7 promoter collection has been used to probe essential gene function in cell size control, cell morphology, mitochondrial morphogenesis, and for gene expression and synthetic genetic interaction profiling (Mnaimneh et al, 2004;Altmann and Westermann, 2005;Davierwala et al, 2005).…”

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A Survey of Essential Gene Function in the Yeast Cell Division Cycle

Peña‐Castillo

Mnaimneh

et al. 2006

MBoC

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Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO 7 promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication. INTRODUCTIONCell division is a fundamental biological process that in all organisms consists of a series of closely coordinated events. In the budding yeast Saccharomyces cerevisiae, the cell division cycle begins with an initial growth phase, G1, during which time the cell increases in mass and volume. The transition from G1 into S phase is marked by progression through Start (the point of commitment to cell division), initiation of nuclear DNA synthesis, and the emergence of a bud that will form the new daughter cell. S phase is followed by a second growth phase, G2, which is in turn followed by nuclear division, and then cell separation. A very similar series of events occurs in all eukaryotes, with the obvious exception of bud emergence. Because of the fundamental biological importance of cell cycle progression and its importance in both human development and diseases such as cancer, identification of the molecular determinants of specific stages of the eukaryotic cell cycle has been a subject of intense study for several decades.The morphological landmarks of the cell cycle stages in budding yeast, most notably the size of the bud relative to the size of the mother cell, allows the identification of mutants blocked at specific stages of the cell cycle and thereby forms the basis for the classic cell cycle screens (Hartwell et al., 1970;Culotti and Hartwell, 1971;Hartwell, 1971aHartwell, , 1971bHartwell, , 1973Moir et al., 1982). These genetic screens, using conditional temperature-sensitive mutants, identified more than 50 genes that are required for specific stages in the cell division cycle and so were termed CDC genes. On average 4.6 alleles were identified for each CDC gene in the original screens, suggesting the number of cdc mutan...

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“…This provides a proof of principle regarding the utility of this approach in generating conditional mutants, distinct from classical methods using temperature-sensitive mutations and expression shut-off approaches (e.g. the use of tetracycline-regulatable promoters) (Mnaimneh et al, 2004). In a conceptually distinct set of experiments, we directed a protein to its site of activity in the absence of native stimuli, driving the calcium-regulated transcription factor Crz1p to the nucleus without corresponding changes in intracellular calcium levels.…”

Section: Discussionmentioning

confidence: 99%

“…With regard to the latter, the yeast genome contains approximately 1130 essential genes, and we readily envision application of our platform to this important yeast gene set. The resulting mutant collection will provide a strong complement to existing genome-wide collections of titratable promoter alleles (Mnaimneh et al, 2004), gene deletion mutants (Winzeler and Davis, 1997;Winzeler et al, 1999) and gene overexpression strains (Sopko et al, 2006;Gelperin et al, 2005). Similarly, genome-scale mislocalization screens will augment pathway discovery tools, such as synthetic genetic array analysis (Tong et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

A small molecule‐directed approach to control protein localization and function

Geda

Patury

et al. 2008

Yeast

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Protein localization is tightly linked with function, such that the subcellular distribution of a protein serves as an important control point regulating activity. Exploiting this regulatory mechanism, we present here a general approach by which protein location, and hence function, may be controlled on demand in the budding yeast. In this system a small molecule, rapamycin, is used to temporarily recruit a strong cellular address signal to the target protein, placing subcellular localization under control of the selective chemical stimulus. The kinetics of this system are rapid: rapamycin-directed nucleo-cytoplasmic transport is evident 10-12 min posttreatment and the process is reversible upon removal of rapamycin. Accordingly, we envision this platform as a promising approach for the systematic construction of conditional loss-of-function mutants. As proof of principle, we used this system to direct nuclear export of the essential heat shock transcription factor Hsf1p, thereby mimicking the cell-cycle arrest phenotype of an hsf1 temperature-sensitive mutant. Our drug-induced localization platform also provides a method by which protein localization can be uncoupled from endogenous cell signalling events, addressing the necessity or sufficiency of a given localization shift for a particular cell process. To illustrate, we directed the nuclear import of the calcineurin-dependent transcription factor Crz1p in the absence of native stimuli; this analysis directly substantiates that nuclear translocation of this protein is insufficient for its transcriptional activity. In total, this technology represents a powerful method for the generation of conditional alleles and directed mislocalization studies in yeast, with potential applicability on a genome-wide scale.

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Exploration of Essential Gene Functions via Titratable Promoter Alleles

Cited by 551 publications

References 59 publications

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

A Survey of Essential Gene Function in the Yeast Cell Division Cycle

A small molecule‐directed approach to control protein localization and function

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