ArticleYeast (Chichester, England)2005
A high-efficiency method to replace essential genes with mutant alleles in yeast.
Article in Yeast (Chichester, England), 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed, 28 citations in OpenAlex.
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- Kingdom-wide CRISPR guide design with ALLEGRO.Nucleic acids research · 2025Article
- Detecting circRNA in purified spliceosomal P complex.Methods (San Diego, Calif.) · 2021Article
- Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae.The Journal of cell biology · 2021Article
- Expansion of the evolutionarily conserved network of J-domain proteins in the Arabidopsis mitochondrial import complex.Plant molecular biology · 2021Article
- Establishing Regulation of a Dynamic Process by Ypt/Rab GTPases : A Case for Cisternal Progression.Methods in molecular biology (Clifton, N.J.) · 2021Article
- Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase.The Journal of biological chemistry · 2020Article
- Cell cycle-dependent association of polo kinase Cdc5 with CENP-A contributes to faithful chromosome segregation in budding yeast.Molecular biology of the cell · 2019Article
- Sumoylation regulates the stability and nuclease activity ofCommunications biology · 2019Article
- Novel phosphorylation states of the yeast spindle pole body.Biology open · 2018Article
- Yeast Still a Beast: Diverse Applications of CRISPR/Cas Editing Technology inThe Yale journal of biology and medicine · 2017Review
- The motor activity of DNA2 functions as an ssDNA translocase to promote DNA end resection.Genes & development · 2017Article
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- Bir1 is required for the tension checkpoint.Molecular biology of the cell · 2009Article
- The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro.Molecular microbiology · 2009Article
- Phosphorylation of the chromosomal passenger protein Bir1 is required for localization of Ndc10 to the spindle during anaphase and full spindle elongation.Molecular biology of the cell · 2006Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
Temperature-sensitive (TS), internally deleted and truncated alleles are important tools to facilitate the characterization of essential genes. We have developed a straightforward method to replace a wild-type gene with a mutant allele at the endogenous locus. This method is an efficient alternative to the two-step method for integration of alleles that are compromised in function or contain multiple mutations. A strain is constructed that has the essential gene of interest disrupted by a selectable marker. Strain viability is maintained by a plasmid carrying a copy of the essential wild-type gene and the ADE3 gene. The mutant allele is cloned into an integratable vector carrying a selectable/counter-selectable marker, such as URA3. The plasmid is linearized and transformed, directing integration to the 5' or 3' region flanking the essential open reading frame (ORF). Transformants that have integrated the mutant gene at the endogenous locus can lose the autonomous plasmid carrying the wild-type copy of the essential gene and the ADE3 gene. These transformants are identifiable as white sectoring colonies, display the mutant phenotype and may be characterized. An optional second selection step on 5-fluoroorotic acid (5-FOA) selects for popouts of the integrating vector sequences, leaves the mutant allele at the endogenous locus, and recycles selectable markers. We have used this method to integrate a TS allele of SPC110 that could not be integrated by standard methods.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.