| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 101 === Maintenance of chromosome ploidy is crucial for sexual reproduction, developmental process and normal function of cells. Several factors controlling chromosome maintenance in Saccharomyces cerevisiae were identified. Among them, Remodeler of the structure of chromatin (RSC) was found. HTL1 (High Temperature Lethal 1) encoding a subunit of RSC which deletion leads to ploidy increase. However, the exact mechanism how HTL1 deletion caused ploidy changes is still unknown. Previous studies in our laboratory showed that the irreversible HTL1-dependent chromosome gain was observed in htl1Δ strains. In order to observe events during Htl1p depletion, a strain containing HTL1 gene driven by galactose inducible promoter which can be manually switch on and off the HTL1 expression, was constructed in our laboratory. After complete depletion of Htl1p, DNA contents gradually shifted from haploid to diploid cells. During this period, chromosome miss-segregation was also observed. In this proposal, I attempt to further study the shift of ploidy in Htl1p-depleted cells. FACS analysis was performed to examine change of DNA content. To monitor DNA content of HTL1 switch-off cells for long period of time, cells are harvested every 24 hours for FACS analysis. Data showed that gradual ploidy shift occurred during this period and fixation at diploid after 6 days. In diploid cells, Htl1p depletion lead to chromosome ploidy shifts from diploid to tetraploid and fixes at tetraploid status. To demonstrate that HTL1-dependent ploidy shift is irreversible and only happens once, HTL1 was re-switched on and further switched off in cells which ploidy have already shifted. Data showed that Htl1p could not affect ploidy change again. To investigate the mechanism of phenomenon of ploidy locked, related conditions are searched. It is known that X chromosome inactivation can be reversible during meiosis, as a consequence, sporulation of ploidy-shifted cells are performed. After sporulation of these tetraploid cells, FACS data showed that DNA content increased again by HTL1 regulation. Taken together, Htl1-dependent ploidy shifts happened once and fixed, furthermore, the mechanism could be reversed during meiosis. In my project, irreversibility of HTL1-dependent ploidy shifts has been demonstrated, furthermore, the relationship between ploidy changes and meiosis was found. It provides a direction dissect the ploidy locked mechanism genetically.
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