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Published on: May 15, 2018
Increased retention of small chromosomes in a novel yeast mutant
1Department of Molecular Bioregulation, Institute of Molecular and Cellular Biology for Pharmaceutical Sciences, Kyoto Pharmaceutical University, Kyoto.
Abstract:
MSC201 and MSC202 are novel yeast mutant strains in which a 10-kb linear yeast artificial chromosome (YAC) was stably maintained in unselective medium during mitotic cell division. After culturing for 35 cycles of cell division, about 50% of MSC cells retain YAC3 DNA, compared to 0.001% of the wild-type AB1380 strain. Southern blot hybridization analysis with pBR322 DNA as the probe showed that in the MSC transformants, YAC DNA remained in a linear form free of cellular chromosomal DNA. The msc201 mutation was shown to be recessive by the rapid loss of the YAC in a diploid strain made by mating with AB1375, which has a genetic background similar to that of the MSC strain. Linear YAC DNA with a centromere was stabilized in MSC201 better than a linear construct lacking a centromere sequence.
Insights
Novel yeast mutant strains, MSC201 and MSC202, demonstrate remarkable stability of linear yeast artificial chromosomes (YACs) during cell division. These strains maintain YAC DNA at significantly higher rates than wild-type strains, offering new possibilities for genetic research.
Area of Science:
- * Molecular Biology
- * Yeast Genetics
- * Chromosome Stability
Background:
- * Maintaining large DNA constructs like yeast artificial chromosomes (YACs) during cell division is a significant challenge in molecular biology.
- * Wild-type yeast strains exhibit poor retention of linear YACs, limiting their utility for genetic studies.
Purpose of the Study:
- * To investigate novel yeast mutant strains (MSC201 and MSC202) for their ability to stably maintain linear YACs.
- * To characterize the genetic basis of YAC stabilization in these mutant strains.
Main Methods:
- * Construction and culturing of yeast mutant strains (MSC201, MSC202) harboring a 10-kb linear YAC.
- * Mitotic cell division tracking over 35 cycles under unselective conditions.
- * Southern blot hybridization to analyze YAC DNA integrity and chromosomal association.
- * Genetic analysis of the msc201 mutation using diploid strains and YACs with/without centromere sequences.
Main Results:
- * MSC strains retained YAC DNA in approximately 50% of cells after 35 divisions, a dramatic increase from 0.001% in wild-type AB1380.
- * YAC DNA in MSC transformants remained linear and unintegrated into chromosomal DNA.
- * The msc201 mutation was recessive, and YAC stabilization was dependent on the presence of a centromere sequence.
Conclusions:
- * MSC201 and MSC202 mutant strains provide a robust system for stable maintenance of linear YACs.
- * The msc201 mutation plays a crucial role in stabilizing linear YACs, particularly those with centromeric elements.
- * These findings have implications for using YACs in yeast-based genetic engineering and functional genomics research.
