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Development of stable haploid strains and molecular genetic tools for Naumovozyma castellii (Saccharomyces castellii)

Karademir Andersson, Ahu LU ; Oredsson, Stina LU and Cohn, Marita LU (2016) In Yeast (Chichester, England) 33(12). p.633-646
Abstract

The budding yeast Naumovozyma castellii (syn. Saccharomyces castellii) has been included in comparative genomics studies, functional analyses of centromere DNA elements, and was shown to possess beneficial traits for telomere biology research. To provide useful tools for molecular genetic approaches, we produced stable haploid heterothallic strains from an early ancestral strain derived from the N. castellii collection strain CBS 4310. To this end, we deleted the gene encoding the Ho endonuclease, which is essential for the mating type switching. Gene replacement of HO with the kanMX3 resistance cassette was performed in diploid strains, followed by sporulation and tetrad microdissection of the haploid spores. The mating type (MATa or... (More)

The budding yeast Naumovozyma castellii (syn. Saccharomyces castellii) has been included in comparative genomics studies, functional analyses of centromere DNA elements, and was shown to possess beneficial traits for telomere biology research. To provide useful tools for molecular genetic approaches, we produced stable haploid heterothallic strains from an early ancestral strain derived from the N. castellii collection strain CBS 4310. To this end, we deleted the gene encoding the Ho endonuclease, which is essential for the mating type switching. Gene replacement of HO with the kanMX3 resistance cassette was performed in diploid strains, followed by sporulation and tetrad microdissection of the haploid spores. The mating type (MATa or MATα) was determined for each hoΔ mutant, and was stable under sporulation inducing conditions, showing that the switching system was totally non-functional. The hoΔ strains showed wild-type growth rates and were successfully transformed with linear DNA using the general protocol. Opposite mating types of the hoΔ strains were mated, resulting in diploid cells that efficiently formed asci and generated viable spores when microdissected. By introduction of a point mutation in the URA3 gene, we created a uracil auxotrophic strain, and by exchanging the kanMX3 cassette for the hphMX4 cassette we show that hygromycin B resistance can be used as a selection marker in N. castellii. These haploid strains containing genetic markers will be useful tools for doing genetic analyses in N. castellii. Moreover, we demonstrate that homology regions of 200-230 bp can be successfully used for target site-specific integration into genomic loci.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Yeast, genetic tools, ho endonuclease, Haploid and diploid yeast, molecular mechanisms, molecular genetics
in
Yeast (Chichester, England)
volume
33
issue
12
pages
14 pages
publisher
John Wiley & Sons
external identifiers
  • scopus:85000428191
ISSN
1097-0061
DOI
10.1002/yea.3213
language
English
LU publication?
yes
id
2f355fbe-1412-4bac-970c-c393c1a059ef
date added to LUP
2016-10-05 18:03:58
date last changed
2017-03-19 04:36:10
@article{2f355fbe-1412-4bac-970c-c393c1a059ef,
  abstract     = {<p>The budding yeast Naumovozyma castellii (syn. Saccharomyces castellii) has been included in comparative genomics studies, functional analyses of centromere DNA elements, and was shown to possess beneficial traits for telomere biology research. To provide useful tools for molecular genetic approaches, we produced stable haploid heterothallic strains from an early ancestral strain derived from the N. castellii collection strain CBS 4310. To this end, we deleted the gene encoding the Ho endonuclease, which is essential for the mating type switching. Gene replacement of HO with the kanMX3 resistance cassette was performed in diploid strains, followed by sporulation and tetrad microdissection of the haploid spores. The mating type (MATa or MATα) was determined for each hoΔ mutant, and was stable under sporulation inducing conditions, showing that the switching system was totally non-functional. The hoΔ strains showed wild-type growth rates and were successfully transformed with linear DNA using the general protocol. Opposite mating types of the hoΔ strains were mated, resulting in diploid cells that efficiently formed asci and generated viable spores when microdissected. By introduction of a point mutation in the URA3 gene, we created a uracil auxotrophic strain, and by exchanging the kanMX3 cassette for the hphMX4 cassette we show that hygromycin B resistance can be used as a selection marker in N. castellii. These haploid strains containing genetic markers will be useful tools for doing genetic analyses in N. castellii. Moreover, we demonstrate that homology regions of 200-230 bp can be successfully used for target site-specific integration into genomic loci.</p>},
  author       = {Karademir Andersson, Ahu and Oredsson, Stina and Cohn, Marita},
  issn         = {1097-0061},
  keyword      = {Yeast,genetic tools,ho endonuclease,Haploid and diploid yeast,molecular mechanisms,molecular genetics},
  language     = {eng},
  month        = {09},
  number       = {12},
  pages        = {633--646},
  publisher    = {John Wiley & Sons},
  series       = {Yeast (Chichester, England)},
  title        = {Development of stable haploid strains and molecular genetic tools for Naumovozyma castellii (Saccharomyces castellii)},
  url          = {http://dx.doi.org/10.1002/yea.3213},
  volume       = {33},
  year         = {2016},
}