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Ends-in vs. ends-out targeted insertion mutagenesis in Saccharomyces castellii.

Astromskas, Eimantas LU and Cohn, Marita LU (2009) In Current Genetics 55(3). p.339-347
Abstract
Gene replacement (knock-out) is a major tool for the analysis of gene function. However, the efficiency of correct targeting varies between species, and is dependent on the structure of the DNA construct. We analyzed the targeted insertion mutagenesis method in the budding yeast Saccharomyces castellii, phylogenetically positioned after the whole genome duplication event in the Saccharomyces lineage. We compared the targeting efficiency for target DNA constructs in the respective ends-in and ends-out form. For some of the constructs S. castellii showed a similar high degree of homologous recombination as S. cerevisiae. In agreement with S. cerevisiae, a higher targeting efficiency was seen for the diploid strain than for the haploid.... (More)
Gene replacement (knock-out) is a major tool for the analysis of gene function. However, the efficiency of correct targeting varies between species, and is dependent on the structure of the DNA construct. We analyzed the targeted insertion mutagenesis method in the budding yeast Saccharomyces castellii, phylogenetically positioned after the whole genome duplication event in the Saccharomyces lineage. We compared the targeting efficiency for target DNA constructs in the respective ends-in and ends-out form. For some of the constructs S. castellii showed a similar high degree of homologous recombination as S. cerevisiae. In agreement with S. cerevisiae, a higher targeting efficiency was seen for the diploid strain than for the haploid. Surprisingly, a higher degree of targeting efficiency was seen for ends-out constructs compared to ends-in constructs. This result may have been influenced by the difference in the length of the homologous target sequences used, although long homology regions of 300 bp-1 kb were used in all constructs. Remarkably, very short regions of cohesive heterologous sequences at the ends of the constructs highly stimulated random illegitimate integration, suggesting that the pathway of non-homologous end joining is highly active in S. castellii. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Current Genetics
volume
55
issue
3
pages
339 - 347
publisher
Springer
external identifiers
  • wos:000267040700010
  • scopus:67349203131
ISSN
0172-8083
DOI
10.1007/s00294-009-0248-8
language
English
LU publication?
yes
id
5c19288b-a82f-4c85-833a-cadc52d75e5a (old id 1412363)
date added to LUP
2009-06-11 09:34:43
date last changed
2017-03-19 03:36:51
@article{5c19288b-a82f-4c85-833a-cadc52d75e5a,
  abstract     = {Gene replacement (knock-out) is a major tool for the analysis of gene function. However, the efficiency of correct targeting varies between species, and is dependent on the structure of the DNA construct. We analyzed the targeted insertion mutagenesis method in the budding yeast Saccharomyces castellii, phylogenetically positioned after the whole genome duplication event in the Saccharomyces lineage. We compared the targeting efficiency for target DNA constructs in the respective ends-in and ends-out form. For some of the constructs S. castellii showed a similar high degree of homologous recombination as S. cerevisiae. In agreement with S. cerevisiae, a higher targeting efficiency was seen for the diploid strain than for the haploid. Surprisingly, a higher degree of targeting efficiency was seen for ends-out constructs compared to ends-in constructs. This result may have been influenced by the difference in the length of the homologous target sequences used, although long homology regions of 300 bp-1 kb were used in all constructs. Remarkably, very short regions of cohesive heterologous sequences at the ends of the constructs highly stimulated random illegitimate integration, suggesting that the pathway of non-homologous end joining is highly active in S. castellii.},
  author       = {Astromskas, Eimantas and Cohn, Marita},
  issn         = {0172-8083},
  language     = {eng},
  number       = {3},
  pages        = {339--347},
  publisher    = {Springer},
  series       = {Current Genetics},
  title        = {Ends-in vs. ends-out targeted insertion mutagenesis in Saccharomyces castellii.},
  url          = {http://dx.doi.org/10.1007/s00294-009-0248-8},
  volume       = {55},
  year         = {2009},
}