X-inactivation in female mammals is triggered by the association of non-coding Xist RNA in cis with the X chromosome. Although it has been suggested that the A-repeat located in the proximal part of the Xist RNA is required for chromosomal silencing in ES cells, its role in mouse has not yet been addressed. Here, we deleted the A-repeat in mouse and studied its effects on X-inactivation during embryogenesis. The deletion, when paternally transmitted, caused a failure of imprinted X-inactivation in the extraembryonic tissues, demonstrating the essential role of the A-repeat in X-inactivation in the mouse embryo. Unexpectedly, the failure of X-inactivation was caused by a lack of Xist RNA rather than by a defect in the silencing function of the mutated RNA, which we expected to be expressed from the mutated X. Interestingly, the normally silent paternal copy of Tsix, which is an antisense negative regulator of Xist, was ectopically activated in the preimplantation embryo. Furthermore, CpG sites in the promoter region of paternal Xist, which are essentially unmethylated in the extraembryonic tissues of the wild-type female embryo, acquire a significant level of methylation on the mutated paternal X. These findings demonstrate that the DNA sequence deleted on the mutated X, most probably the A-repeat, is essential as a genomic element for the appropriate transcriptional regulation of the Xist/Tsix loci and subsequent X-inactivation in the mouse embryo.

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In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, are discussed.

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