Naslov
Preferential DNA sequences 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
YCGI - Yeast Cooperation Group in Italy 2000, Book of abstracts / - Cortona, 2000

Skup
YCGI - Yeast Cooperation Group in Italy 2000

Mjesto i datum
Cortona, Italija, 22.04.2000. - 25.04.2000

Vrsta sudjelovanja
Pozvano predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
yeast; gene targeting; hot spots; mismatch repair

Sažetak
Compared to gene deletion methodologies in higher eukaryotes, current gene knock out in yeast is impressively successful. However, the efficiency of gene targeting in this microorganism 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 their relevant features are, 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 discontinous gene-like sequences named "POT" (P: promoter, O: ORF, T: terminator). Plasmid DNA, linearized by restriction enzymes within one of the three gene regions, was used to transform the haploid yeast strain S288C by integrating into its homologous DNA 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 Southern blot analysis to verify the DNA site of integration. It was found that plasmid targeting into the ORF region of all three genes was highly unsuccessful. Indeed, a large portion of the transformants investigated (42% for URA3, 51% for HIS3 and 48% for TRP1), obtained with plasmids linearized within the ORF region, contained the plasmid DNA integrated into one of the other two regions P and T. By comparing the frequency of these mis-targeting events, promoter and terminator-containing DNA sequences were found to be preferred for gene targeting up to 10 times more than their corresponding ORF. The same type of experiment was performed with plasmids containing only two functional gene regions of the URA3 (pURA-PO and pURA-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 competence between ORFs, and neighboring DNA sequences. Furthermore, by using isogenic knockout yeast strains deleted for some of the mismatch-repair genes, we demonstrated the involvement of these gene products in the process of the gene targeting. For instance, a further decrease in correct targeted plasmid integration into the URA3 ORF was noticed in the delta msh3 and even more significantly in the delta msh2 null strains, was only about 20% of cases plasmid DNA was successfully targeted into the proper URA3 ORF region.

Izvorni jezik
Engleski

POVEZANOST RADA