In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.

The mdm2 oncogene encodes a 90-kDa protein that can bind to the p53 tumor suppressor protein and negatively regulate its functions in transcription, cell cycle arrest, and apoptosis. The mdm2 gene is frequently amplified in human sarcomas, which may be responsible for the malignant transformations. We present evidence that the mdm2 oncoprotein is cleaved by an interleukin 1␤-converting enzyme-like protease (caspase) during p53-mediated apoptosis. The protease that cleaves mdm2 has a specificity similar to that of CPP32 (caspase-3), and recombinant caspase-3 is able to cleave mdm2 in vitro. The protease cleavage site has been mapped to between residue 361 and 362 of human mdm2. The proteolytic cleavage removes the COOH-terminal RING finger domain of mdm2, resulting in the loss of RNA binding activity. The p53 binding and inhibition functions of mdm2 are not affected by the cleavage. The cleavage site sequence of mdm2 is evolutionarily conserved, suggesting that regulation by caspase cleavage during apoptosis is an important feature of mdm2.

XCoe2 may play a pivotal role in the transcriptional cascade that specifies primary neurons in Xenopus embryos: by maintaining Delta-Notch signalling, XCoe2 stabilises the higher neural potential of selected progenitor cells that express X-ngnr-1, ensuring the transition between neural competence and irreversible commitment to a neural fate; and it promotes neuronal differentiation by activating XNeuroD expression, directly or indirectly.

For the success of fertilization, spindles of vertebrate oocytes must remain stable and correctly organized during the arrest in metaphase II of meiosis. Using a two-hybrid screen with MAPK as a bait, we have recently identified MISS (MAPK interacting and spindle stabilizing) which controls mouse oocyte metaphase II spindle stability. Using the same screen, we identify another MAPK partner, DOC1R (Deleted in oral cancer one related), a murine homologue of a potential human tumor suppressor gene. We characterize DOC1R during mouse oocyte meiosis resumption. DOC1R is regulated by phosphorylation during meiotic maturation by MPF (M-phase promoting factor)and by the MOS/.../MAPK pathway. DOC1R and a DOC1R-GFP fusion localize to microtubules during meiotic maturation. Consistent with this microtubular localization, we show, by antisense and double-stranded RNA injection, that depletion of DOC1R induces microtubule defects in metaphase II oocytes. These defects are rescued by overexpressing a Xenopus DOC1R, showing that they are specific to DOC1R. Thus, the discovery of DOC1R, a substrate of MAPK that regulates microtubule organization of metaphase II mouse oocytes, reinforces the importance of this pathway in the control of spindle stability during the metaphase II arrest.

During vertebrate oogenesis and early embryogenesis, gene expression is governed mainly by translational control. The recruitment of Poly(A) Binding Protein (PABP) during poly(A) tail lengthening appears to be the key to translational activation during this period of development in Xenopus laevis. We showed that PABP1 and ePABP proteins are both present during oogenesis and early development. We selected ePABP as an eRF3 binding protein in a two-hybrid screening of a X. laevis cDNA library and demonstrated that this protein is associated with translational complexes. It can complement essential functions of the yeast homologue Pab1p. We discuss specific expression patterns of the finely tuned PABP1 and ePABP proteins.

We have isolated from a subtractive cDNA library of Xenopus laevis a novel transcript, H2A.ZI, which belongs to the H2A.Z variant gene family. Characterization of its expression during oogenesis and development shows significant differences from the expression of the core histone H2A. First, H2A.ZI mRNA is mainly detected only during oogenesis and after the midblastula transition, whereas H2A is constitutively expressed, at much higher levels, throughout embryonic growth. Second, in contrast with H2A, the variant H2A.ZI is polyadenylated during development. Third, expression of H2A.ZI is uncoupled from the S phase after gastrula, whereas synthesis of the core histone H2A mRNA is tightly controlled to DNA replication. Interestingly, H2A.ZI is less charged in the N-terminal tail which is crucial for chromatin-mediated repression. The characteristics of H2A.ZI suggest that its incorporation into nucleosomes would lead to a chromatin structure more competent for gene expression during development.

SUMMARYAs a preliminary step towards elucidation of the molecular basis of ectomycorrhiza differentiation, polypeptide changes at different stages of mycorrhiza development were analyzed in birch {Betula pendula Roth). Timesequencing of the stages in the infection process of clonal plants inoculated with a compatible isolate of Paxillus involutus Batsch revealed that by 8 d mature ectomycorrhizas were obtained. Total phenol-extracted proteins of roots during the ' mycorrhiza formation stage' (2-8 d post-inoculation) were separated by two-dimensional polyacrylamide gel electrophoresis and the resulting patterns compared with non-mycorrhizal roots and mycelium. Alteration in the concentration of polypeptides from the host plant roots was limited even after 8 d of contact between the symbionts. However, seven novel polypeptides were detected 4 d after inoculation, three of them being already present m 2-d-old ectomycorrhizas. These findings demonstrate that symbiosis-related polypeptides accumulate in ectomycorrhizal roots prior to any of the morphological changes characterizing the symbiotic state.

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