Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha

K N Faber, G J Swaving, F Faber, G Ab, W Harder, M Veenhuis, P Haima

Onderzoeksoutput: ArticleAcademicpeer review

Uittreksel

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

Originele taal-2English
Pagina's (van-tot)2405-2416
TijdschriftJournal of general microbiology
Volume138
Nummer van het tijdschrift11
DOI's
StatusPublished - nov 1992

Keywords

  • genetica
  • dna

Citeer dit

Faber, K N ; Swaving, G J ; Faber, F ; Ab, G ; Harder, W ; Veenhuis, M ; Haima, P. / Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha. In: Journal of general microbiology. 1992 ; Vol. 138, Nr. 11. blz. 2405-2416.
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abstract = "Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22{\%} of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68{\%} of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.",
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author = "Faber, {K N} and Swaving, {G J} and F Faber and G Ab and W Harder and M Veenhuis and P Haima",
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Faber, KN, Swaving, GJ, Faber, F, Ab, G, Harder, W, Veenhuis, M & Haima, P 1992, 'Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha' Journal of general microbiology, vol. 138, nr. 11, blz. 2405-2416. https://doi.org/10.1099/00221287-138-11-2405

Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha. / Faber, K N; Swaving, G J; Faber, F; Ab, G; Harder, W; Veenhuis, M; Haima, P.

In: Journal of general microbiology, Vol. 138, Nr. 11, 11.1992, blz. 2405-2416.

Onderzoeksoutput: ArticleAcademicpeer review

TY - JOUR

T1 - Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha

AU - Faber, K N

AU - Swaving, G J

AU - Faber, F

AU - Ab, G

AU - Harder, W

AU - Veenhuis, M

AU - Haima, P

PY - 1992/11

Y1 - 1992/11

N2 - Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

AB - Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

KW - dna

KW - genetic engineering

KW - genome, fungal

KW - plasmids/genetics

KW - recombination, genetic

KW - transformation, genetic

KW - genetica

KW - dna

U2 - 10.1099/00221287-138-11-2405

DO - 10.1099/00221287-138-11-2405

M3 - Article

VL - 138

SP - 2405

EP - 2416

JO - Journal of general microbiology

JF - Journal of general microbiology

SN - 0022-1287

IS - 11

ER -