Naslov
Specific chromosome loss induction by centromere knockout in Saccharomyces cerevisiae followed by restitution of homozygous diploidy

Autori
Zang, Yuhui ; Garre, Massimiliano ; Gjuračić, Krešimir ; Bruschi, Carlo V.

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
20th International Conference on Yeast Genetics and Molecular Biology, Yeast, Special Conference Issue / - Prag : Willey & Sons, 2001, 6-10

Skup
20th International Conference on Yeast Genetics and Molecular Biology

Mjesto i datum
Prag, Češka Republika, 26.08.2001. - 31.08.2001

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
yeast; knockout; centromere; restituition of diploidy

Sažetak
To investigate the possibility of inducing specific chromosome loss by centromere deletion in eukaryotic cells, the yeast diploid strain ZG1, carrying three pairs of heterozygous markers (CAN1S/can1R, URA3/ura3, hphMX4/HIS1) widely spread on both arms of chromosome V was constructed. One of the two centromeres V of ZG1 was replaced by the LEU2 gene via the PCR-mediated knockout technique. After DNA transformation, putative yeast colonies that showed loss of heterozygosity (LOH) for the three markers of chromosome V were identified among the colonies selected for leucine prototrophy. Phenotypic tests, colony-PCR and Southern-blot analysis of these cells demonstrated the physical loss of the Can1s, URA3, and hphMX4 marker genes from the genome. Further tetrad analysis results were consistent with this conclusion ; however, four-spore viability indicated a normal chromosome number of these transformants. To verify the diploidy of the selected chromosome V, the HIS1 gene was deleted with a standard KanMX4 knockout DNA cassette. The resulting heterogeneity of the HIS1/KanMX4 markers, together with further quantitative PCR and densitometric analysis on chromosome V, confirmed its diploid complement, thereby indicating that an endoreduplication event had taken place. These results demonstrate that it is possible to manipulate simultaneously the genomic configuration of several linked genetic markers by specifically interfering with the stability and transmission of their chromosome

Izvorni jezik
Engleski

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