title: PCR-based gene disruption and recombinatory marker excision to produce modified industrial Saccharomyces cerevisiae without added sequences.
abstract: The dominant selectable Kanr marker, which confers geneticin resistance in yeast, is extensively used for PCR based disruption of genes in functional analysis studies in laboratory strains of Saccharomyces cerevisiae. We have developed a gene disruption cassette, which incorporates the Kanr marker, and direct repeat sequences designed from the target gene to enable the deletion of the gene without the introduction of added DNA sequences. We report on the disruption of the HO gene as a test case, using the hodr-Kanr-hodr cassette. The cassette was shown to integrate at the HO locus and the Kanr marker excised by recombination between the two direct repeat sequences. The disruption/excision event resulted in the removal of one direct repeat and the coding sequence of the gene, and hence in this case loss of HO function, with the introduction of no foreign or additional sequences, including the Kanr marker. Having been derived from the target site, the remaining direct repeat sequence is native sequence in its native location. This design template has the potential to be adapted to other genes, and as such will be of advantage in instances such as the optimization of strains by recombinant DNA technology where the retention of minimal or no foreign sequences is desired.
authors.affiliation: School of Agriculture and Wine, The University of Adelaide, PMB 1 Glen Osmond, Adelaide, SA 5064, Australia. michelle.walker@adelaide.edu.au
authors.forename: Michelle
authors.initials: M
authors.lastname: Walker
authors.forename: Andrea
authors.initials: A
authors.lastname: Vystavelova
authors.forename: Scott
authors.initials: S
authors.lastname: Pedler
authors.forename: Jeff
authors.initials: J
authors.lastname: Eglinton
authors.forename: Vladimir
authors.initials: V
authors.lastname: Jiranek
mesh_terms: D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D005819:Genetic Markers; D005839:Gentamicins / Q000494:pharmacology; D007218:Industrial Microbiology / Q000379:methods*; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014920:Wine / Q000382:microbiology*
chemical_list: D005819:Genetic Markers; D005839:Gentamicins; D029701:Saccharomyces cerevisiae Proteins; C010680:antibiotic G 418; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific