Chromosome V loss due to centromere knockout or MAD2-deletion is immediately followed by restitution of homozygous diploidy in Saccharomyces cerevisiae

Yeast
Yuhui ZangCarlo V Bruschi

Abstract

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 marker genes (CAN1(S)/can1(R), URA3/Deltaura3, 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 well-established PCR-mediated knockout technique. After DNA transformation, putative yeast colonies that showed loss of heterozygosity (LOH) for the three markers of chromosome V (CAN1(S) URA3 hphMX4) 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 CAN1(s), 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 quantitative PCR and densitometric analysis on chromosome V, confirmed its diploi...Continue Reading

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Citations

Dec 23, 2008·Mutation Research·Xiaowei WuMarek Kimmel
Jul 22, 2005·Yeast·Sanjeev K Waghmare, Carlo V Bruschi
Dec 17, 2005·Yeast·Jesús AmbronaManuel Ramírez
Jul 12, 2002·Yeast
Aug 19, 2007·Science·Prasad V Jallepalli, David Pellman

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