Mammalian cell cycle regulation : characterisation of three genes expressed in proliferating cells

Proliferating cells undergo different phases in their life cycle. During interphase the cells grow and duplicate their DNA content, followed by a cell division phase, mitosis, which results in two daughter cells. The transitions between the different events of the cell cycle are strictly regulated by a checkpoint machinery to ensure the integrity of the DNA.This control system checks that an early event is completed before a later event is initiated. For example, the DNA replication must be completed before a cell can enter mitosis. The failure of any of the checkpoints could lead to uncontrolled cell proliferation and the accumulation of genetic damage. It is therefore of great interest to identify and characterise new genes that are involved in the control of the cell cycle.

This thesis presents the isolation and characterisation of three murine genes, tsg23, tsg24 and tsgl26, all expressed in proliferating cells. The first gene described, tsg23, was shown to be the murine homologue, Pl, of the yeast MCM3 gene, which has been shown to be important for the initiation of DNA replication. The MCM3 protein belongs to a protein family suggested to function as replication licensing factors. The Pl protein is located in the nuclei during all stages of interphase and in the extrachromosomal regions during mitosis. The P1 protein accumulates in the heterochromatin regions before replication, an association which is lost during the Sphase. Our results suggest that the Pl protein could be involved in the regulation of the initiation of DNA replication. In contrast to MCM3, we found that P1 remains localised in the nuclei throughout interphase. This rises the possibility that the accumulation of the mammalian MCM proteins are regulated by a different mechanism than theirs yeast counterparts. An alternative explanation is that the activity of the yeast MCM proteins could be regulated by two different mechanisms.

The second gene described, tsg24, possibly represents the murine homologue of the Aspergillus nidulans BimE gene. It has been suggested that the BimE protein functions as acell cycle checkpoint regulator, which controls the transition between different checkpoints.The Tsg24 protein is a nuclear protein located in the centromere regions of the chromosomes, chromosomal regions important for the proper segregation of sister chromatids during mitosis. The centromeric localisation of the Tsg24 protein most probably explains one of the observed BimE phenotypes, the metaphase arrest, as inactivation of other centromere proteins also leads to a metaphase arrest. More intriguing is the second phenotype displayed by BimE mutants, premature chromosome condensation. However, it is possible that some centromere proteins also may be involved in the regulation of chromosome condensation. Interestingly, another centromere protein, CENP-D, is related to RCC1, a protein also known to regulate chromosome condensation.

The third gene investigated, tsgl26, was found to be the murine homologue of the human Ki-67 gene. The Ki-67 protein is exclusively expressed in proliferating cells and is therefore used as a clinical marker to assess the proliferative capacity of tumour cells. Both the Tsgl26 and Ki-67 proteins contain repetitive sequences containing several conserved motifs, including one motif observed in proteins which interact with DNA. The Tsgl26 protein begins to accumulate in the nucleus at the beginning of the G1 phase. During the Sand the G2 phases, the Tsgl26 protein becomes localised to the nucleolus and to the heterochromatin regions. At the onset of mitosis, the Tsgl26 protein is redistributed and associates with the mitotic chromosomes. The intracellular distribution of the Tsgl26 protein during the cell cycle suggests that it could have a chromatin-associated function in both interphase and mitotic cells. Microinjection of anti-Tsgl26 antibodies into proliferating cells delays cell cycle progression, indicating that the Tsgl26 protein has an essential nuclear function.