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内容記述 |
Irrespective of micro organisms or metazoans, cellular components sometimes unevenly inherited to the progenies during cell division. Currently, this phenomenon, which is known as an asymmetric cell division, is shown to be intimately connected with development and cellular homeostasis maintenance. In the asymmetric cell division subcellular constituents including transcripts, proteins, and organelle such as endoplasmic reticulum and centriole segregate unevenly. Moreover, chromosomal DNAs are also inherited unevenly, though the phenomenon remains to be confirmed. In this study, I constructed an assay system that enables us to detect the non-random sister chromatids segregation. Using the system I analyzed if the event is taken place in the budding yeast <i>Saccharomyces cerevisiae</i>. As a result, I succeeded in obtaining evidence that two sisters are selected and non-randomly segregated.<br /> As a model system to analyze the non-random chromosome segregation, I employed the rDNA repeats that reside on the chromosome XII in <i>S. cerevisiae</i>. In the region, the copy number of rDNA repeats frequently varies and it was expected that distinct sister chromatids arise during DNA replication. For this reason, I assumed the non-random sister chromatid segregation can be observed in the rDNA. I continuously separated the progenies of cell division (the daughter and mother cells) by centrifugal elutriation to trace the fate of sister chromatids that are bearing the rDNA repeats. Consequently, when compared the rDNA copy number in the sorted cells, the daughter and mother cell lineages clearly differed. The daughter lineage constituently inherited a sister chromatid that harbors increased copy number of rDNA, while in the mother lineage the number did not change. Therefore, it seemed like that the Chr. XII was differentiated during cell division and non-randomly segregated to the progenies. Remarkably, the pattern of sister chromatids inheritance showed the lineage specificity. This indicated that <i>S. cerevisiae</i> was recognizing the two sister chromatids.<br /> To explore the <i>cis</i> acting mechanisms underlying the non-random sister chromatid segregation, I analyzed the effect of centromere. In <i>S. cerevisiae</i>, centromeric sequences that associate with the kinetochore proteins have directionality. And also, some of the mitotic apparatuses involved in chromosome segregation are polarized during cell division. I speculated that there are some relationship between the polarity of centromere and mitotic apparatus, and the non-random sister chromatid segregation. To analyze the relationship, I exchanged the <i>CEN12</i>(Chr. XII centromere) sequences with other centromeric fragments that harbors opposite directionality and analyzed the phenotypes in the strain. Although the pattern of sister chromatid segregation was anaryzed in the <i>CEN12</i>-modified strains, the directionality of centromere seemed not to be important.<br /> As the other case, I also analyzed the effect of the directionality of rDNA repeat. On the tandemly aligned rDNA repeats, several biological processes, including transcription and replication, are performed in unidirectional way. I investigated whether this directionality is involved in the asymmetric chromosome segregation. For this purpose, the rDNA repeat was reconstructed in inverted direction using a strain that lost the rDNA repeat completely. Unexpectedly, the newly introduced rDNA repeats had lost the competency for increasing their copy number on Chr. XII. Therefore, the effect of the rDNA directionality could not be estimated.<br /> Apart from the <i>cis</i> elements, then I wondered if <i>trans</i> factors associate with the regulation of the sister chromatid segregation. In the mutants that affect stability(<i>sir2</i>△),nuclear localization (<i>heh1</i>△), and segregation (<i>bud6</i>△) of the IDNA, the fate of sister chromatid segtegation was analyzed. In the <i>sir2</i>△ and <i>heh1△</i> the segregation pattern of Chr. XII was equivalent to that of WT.In the <i>bud6</i>△,it was not able to detect the rDNA copy number change by our analysis. From these result, I speculated that nuclear positioning and chromatin stnrctures of the rDNA had little to do with the pattern of sister chromatid segregation.<br /> Finally, I concerned about the possibility that the non-random chromosome segregation specifrcally occurred in the Chr. XII. To investigate this possibility. I performed BrdU pulse-chase analysis to trace the segregation pattern of whole chromosomes. In this analysis, I could not observe the apparently biased DNA strand retention in 16 chromosomes including the Chr. XII at least when recombination in the rDNA was repressed.<br /> In conclusion, I obtained the first evidence of the non-random chromosome segregation in <i>S. cerevisiae.</i> Thus <i>S. cerevisiae</i> seems to maintains a system which distinguish the two sister chromatids during cytokinesis. Further study will reveal how such a chromosome segregation was taken place |
要旨・審査要旨 (331.5 kB)
