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Updated: Jul 28, 2026
08:48
Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast
Published on: January 26, 2017
Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational
K Gjuracic1, E Pivetta, C V Bruschi
1Microbiology Group, ICGEB, Area Science Park, Padriciano 99, 34012 Trieste, Italy.
Molecular Genetics and Genomics : MGG
|March 30, 2004
Summary
Gene targeting in yeast shows promoter and terminator regions are preferred for plasmid integration over coding sequences (ORFs). Coding sequences act as "cold spots," with frequent mis-targeting observed during gene targeting experiments.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Gene Targeting
Background:
- Gene targeting is crucial for genetic manipulation in yeast.
- Understanding DNA integration preferences is key for efficient gene editing.
Purpose of the Study:
- To investigate the efficiency of gene targeting within different DNA segments (promoter, ORF, terminator) in yeast.
- To identify preferred integration sites for double-stranded plasmids in yeast.
Main Methods:
- Transformation of yeast cells with integrative plasmids carrying functional gene domains (promoter, ORF, terminator).
- Analysis of plasmid integration sites using common genetic markers (HIS3, LEU2, TRP1, URA3).
- Linearization of plasmids to target specific integration sites.
Main Results:
- Promoter (P) and terminator (T) regions were preferred integration sites over open reading frames (ORFs) in HIS3 and URA3 genes.
- Linearized plasmids showed ORFs were less preferred integration sites than flanking regions.
- Up to 50% of transformants with plasmids linearized within ORFs integrated into neighboring regions, indicating ORF mis-targeting.
Conclusions:
- Yeast coding sequences (ORFs) are significantly less preferred for plasmid integration.
- Coding sequences function as "cold spots" for plasmid integration in yeast.
- ORF mis-targeting is not due to competition with distal homologous DNA regions.
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