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
HB2000 Book of abstracts / - Zagreb, 2000

Skup
Annual Meeting of Croatian Biochemists with international participation HB2000

Mjesto i datum
Zagreb, Hrvatska, 13.10.2000. - 15.10.2000

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
yeast; gene targeting; hot spots

Sažetak
Compared to gene deletion methodologies in higher eukaryotes, current gene knockout in yeast is impressively successful. However, the efficiency of gene targeting in yeast varies with targeted DNA sequences, and seems to be influenced by different chromatin organization, transcription level, and replication, without evident rules. To better understand why some DNA regions are more recombinagenic among others, and which are their relevant features, we have designed an experimental system based on yeast transformation with non-replicative, integrating linear plasmids, containing part of the functional regions of the genes URA3, HIS3 and TRP1. For each gene, partial DNA fragments of similar size, deriving from their promoter, ORF and terminator sequences, were amplified by PCR and subcloned into the plasmid vector pUG7kanMX4, to form discontinuous gene-like sequences named "POT" (P: promoter, O: ORF, T: terminator). Plasmid DNA, linearised with restriction enzymes within one of the three gene regions, was used to transform the haploid yeast strain S288C by integrating into its homologous DAN region of the wild-type copy of the gene. The efficiency of plasmid targeting was estimated by replica-plating individual geneticin (G418)-resistant transformants onto minimal selective media followed by colony-PCR to verify the 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: 47, 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. The same type of experiment was performed with plasmids containing only two functional DNA regions of the URA3 gene (pURA3-PO and pURA3-OT). The obtained frequencies of mis-targeting into the URA3 ORF with these two plasmids were almost as high as the one for the plasmid pURA-POT, suggesting that this result could not be the consequence of simple homology competition between ORFs and neighbouring DNA sequences. The influence of the transcription process onto plasmid integration was tested with isogenic strain S288C-UPOT containing non-transcribed ura3::POT locus and the plasmid pURA3KAN bearing the functional URA3 gene. Efficiencies of plasmid targeting into P- and T-URA3 domains did not differ significantly from ones obtained with the wild type strain S288C, suggesting that preferential plasmid targeting isn't triggered by transcription process itself. Furthermore, by using isogenic yeast strain deleted for some of the mismatch-repair genes, we demonstrated the involvement of these gene products in the process of gene targeting. For instance, a further decrease in correct targeted plasmid integration into the URA3 ORF was noticed in the Δ msh3 and even more significantly in the Δ msh2 null mutant strains, where in only about of 20% of cases plasmid DNA was successfully targeted into the proper URA3 ORF region.

Izvorni jezik
Engleski

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