Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast

Gray, Misa; Kupiec, Martin; Honigberg, Saul M.

doi:10.1186/1472-6750-4-7

Cited by 24 publications

(17 citation statements)

References 32 publications

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“…To generate a yeast strain with point mutations in Num1 CC (L167E L170E; Fig. 2 A ), we used the site-specific genomic mutagenesis approach ( Gray et al, 2004 ). In brief, after integration of the URA3 cassette into the num1 CC locus (replacing nucleotides 499–510, corresponding to amino acids L167–L170), a PCR product amplified from pSM37 (see Plasmid construction) containing the desired nucleotide substitutions was transformed into the URA3 -integrated strain and subsequently selected on 5-fluoroorotic acid–containing plates.…”

Section: Methodsmentioning

confidence: 99%

The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

Lammers

Markus

2015

Journal of Cell Biology

103

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Section: Methodsmentioning

confidence: 99%

The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

Lammers

Markus

2015

Journal of Cell Biology

103

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“…The construction was done by PCR-based mutagenesis involving two sequential steps (Gray et al 2004). The gene of interest was first replaced by an URA3 cassette with ∼45 bp flanking homologous regions to the gene of interest at both ends.…”

Section: Allele Quantification With Pyrosequencingmentioning

confidence: 99%

Expression evolution in yeast genes of single-input modules is mainly due to changes in trans-acting factors

Wang¹,

Sung²,

Wang³

et al. 2007

Genome Res.

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Both cis-and trans-regulatory mutations contribute to gene expression divergence within and between species. To estimate their relative contributions, we examined two yeast strains, BY (a laboratory strain) and RM (a wild strain), for their gene-expression divergence by microarray. Using these data and published ChIP-chip data, we obtained a set of single-regulator-regulated genes that showed expression divergence between BY and RM. We randomly selected 50 of these genes for further study. We developed a step-by-step approach to assess the relative contributions of cisand trans-variations to expression divergence by using pyrosequencing to quantify the mRNA levels of the BY and RM alleles in the same culture (co-culture) and in hybrid diploids. Forty genes showed expression divergence between the two strains in co-culture, and pyrosequencing of the BY/RM hybrid diploids showed that 45% (18/40) can be attributed to differences in trans-acting factors alone, 17.5% (7/40) mainly to trans-variations, 20% (8/40) to both cis-and trans-acting factors, 7.5% (3/40) mainly to cis-variations, and 10% (4/40) to cis-acting factors alone. In addition, we replaced the BY promoter by the RM promoter in each of 10 BY genes that were found from our microarray data to have expression divergence between BY and RM, and in each case our quantitative PCR analysis revealed a cis effect of the promoter replacement on gene expression. In summary, our study suggests that trans-acting factors play the major role in expression evolution between yeast strains, but the role of cis variation is also important.

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“…Because this recombination occurs at very low frequencies, the resulting ura3Δ isolates are identified by initially selecting for co-transformation with a TRP1 plasmid and then replica-plating hundreds of these transformants to FOA medium [14]. This protocol uses many plates and is relatively work-intensive, so we asked whether substituting URA3 with the CORE-GS cassette, which includes the Gal-SceI fusion gene and the unique SceI site as well as URA3 (see Background), would improve the efficiency of targeted mutagenesis.…”

Section: Resultsmentioning

confidence: 99%

GAL1-SceI directed site-specific genomic (gsSSG) mutagenesis: a method for precisely targeting point mutations in S. cerevisiae

et al. 2011

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BackgroundPrecise targeted mutations are defined as targeted mutations that do not require the retention of other genetic changes, such as marker genes, near the mutation site. In the yeast, S. cerevisiae, there are several methods for introducing precise targeted mutations, all of which depend on inserting both a counter-selectable marker and DNA bearing the mutation. For example, the marker can first be inserted, and then replaced with either a long oligonucleotide carrying the mutation (delitto perfetto) or a PCR fragment synthesized with one primer containing the mutation (SSG mutagenesis).ResultsA hybrid method for targeting precise mutation into the genomes uses PCR fragments as in SSG mutagenesis together with a CORE cassette devised for delitto perfetto that contains the homing endonuclease SceI. This method, termed gsSSG mutagenesis, is much more efficient than standard SSG mutagenesis, allowing replacements to be identified without extensive screening of isolates. In gsSSG, recombination between the PCR fragment and the genome occurs equally efficiently regardless of the size of the fragment or the distance between the fragment end and the site of marker insertion. In contrast, the efficiency of incorporating targeted mutations by this method increases as the distance between the mutation and the marker insertion site decreases.ConclusiongsSSG is an efficient way of introducing precise mutations into the genome of S. cerevisiae. The frequency of incorporating the targeted mutation remains efficient at least as far as 460 bp from the insertion site meaning that a single insertion can be used to create many different mutants. The overall efficiency of gsSSG can be estimated based on the distance between the mutation and the marker insertion, and this efficiency can be maximized by limiting the number of untargeted mutations. Thus, a single insertion of marker genes plus homing endonuclease cassette can be used to efficiently introduce precise point mutations through a region of > 900 bp.

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Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast

Cited by 24 publications

References 32 publications

The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

Expression evolution in yeast genes of single-input modules is mainly due to changes in trans-acting factors

GAL1-SceI directed site-specific genomic (gsSSG) mutagenesis: a method for precisely targeting point mutations in S. cerevisiae

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