title: Development of a system for multicopy gene integration in Saccharomyces cerevisiae.
abstract: In this study we describe construction and evaluation of a vector for multicopy integration in yeast Saccharomyces cerevisiae. In this vector a modified selective marker and a reporter gene PHO8 (encoding alkaline phosphatase) were flanked with delta sequences of the Ty1 transposon. Modified by error-prone PCR version of selection marker kanMX4 was obtained from Escherichia coli clone with impaired geneticin (G418) resistance. The attenuation of kanMX4 gene provides an opportunity to select for explicitly multicopy integration of the module in S. cerevisiae using moderate (200 mg L(-1)) antibiotic concentrations. The developed system provided integration of 3-10 copies of the module in the genome of S. cerevisiae. High copy integration events were confirmed by qRT-PCR, Southern hybridization and reporter enzyme activity measurements.
authors.affiliation: Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine.
authors.forename: Marta V
authors.initials: MV
authors.lastname: Semkiv
authors.affiliation: Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine.
authors.forename: Kostyantyn V
authors.initials: KV
authors.lastname: Dmytruk
authors.affiliation: Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine; University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601, Poland. Electronic address: sibirny@cellbiol.lviv.ua.
authors.forename: Andriy A
authors.initials: AA
authors.lastname: Sibirny
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018244:Chromosomes, Artificial, Yeast; D004352:Drug Resistance, Microbial; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005784:Gene Amplification; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D005839:Gentamicins; D029701:Saccharomyces cerevisiae Proteins; C010680:antibiotic G 418; D000469:Alkaline Phosphatase; C510355:PHO8 protein, S cerevisiae
keywords: Alkaline phosphatase; Geneticin resistance; Multicopy integration; Saccharomyces cerevisiae