Pregled bibliografske jedinice broj: 147387
Preferential DNA regions for gene targeting in the yeast Saccharomyces cerevisiae
Preferential DNA regions for gene targeting in the yeast Saccharomyces cerevisiae // Yeast Genetics and Molecular Biology 2000, Book of abstracts
Seattle (WA), 2000. (poster, međunarodna recenzija, sažetak, znanstveni)
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Naslov
Preferential DNA regions for gene targeting in the yeast Saccharomyces cerevisiae
Autori
Gjuračić, Krešimir ; Bruschi, Carlo V.
Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni
Izvornik
Yeast Genetics and Molecular Biology 2000, Book of abstracts
/ - Seattle (WA), 2000
Skup
Yeast Genetics and Molecular Biology 2000
Mjesto i datum
Seattle (WA), Sjedinjene Američke Države, 25.07.2000. - 30.07.2000
Vrsta sudjelovanja
Poster
Vrsta recenzije
Međunarodna recenzija
Ključne riječi
yeast; gene targeting; hot spots
Sažetak
We transformed yeast cells with non-replicative, integrating linear plasmids containing portions of the genes URA3, HIS3 and TRP1. For each gene, partial DNA fragments of similar size, deriving from their promoter (P), ORF (O) and terminator (T) sequences, were cloned into plasmid pUG7kanMX4, to form discontinuous, gene-like POT sequences. Plasmid DNA, linearised within one of these three gene regions, was used to transform strain S288C by integration into the homologous sequence of the corresponding wild-type gene. Efficiency of plasmid targeting was estimated by replica-plating individual geneticin (G418)-resistant transformants onto selective media, followed by PCR and Southern blot analysis to verify the exact DNA site of integration. While DNA integration into the P and T regions was very efficient, targeting into the ORF region of all three genes was highly unsuccessful. 42, 51 and 48% of the transformants obtained with plasmids linearised within the ORF region of the three target genes respectively, contained the transforming DNA integrated into P or T. Promoter and terminator DNA sequences were found to be preferred for gene targeting up to 10 times more than their corresponding ORF. A further decrease in correct targeted plasmid integration into the URA3 ORF was noticed in the  msh3 and even more significantly in the  msh2 null strains, where in only about 20% of cases plasmid DNA was successfully targeted into the proper URA3 ORF region.
Izvorni jezik
Engleski
