Elisabeth Mandart

c-Mos and cyclin B/cdc2 connections during Xenopus oocyte maturation

Summry— Fully‐grown G2 arrested Xenopus oocytes can be induced to enter and progress into meiotic cell cycle by progesterone stimulation. This process is termed oocyte maturation. An early response to progesterone is the synthesis of the onco‐protein c‐Mos, defined as the candidate initiator of Xenopus oocyte maturation, which triggers the MAPK cascade, MPF activation and promotes CSF activity. Here we review our current knowledge on the synthesis, activation and functions of c‐Mos in connection with MPF activation during maturation. We also discuss our recent results concerning the dispensability of cyclin B degradation in meiosis I‐meiosis II transition and the stabilization of c‐Mos through its direct phosphorylation by cyclin B/cdc2.

SLS1, a newSaccharomyces cerevisiae gene involved in mitochondrial metabolism, isolated as a syntheticlethal in association with anSSM4 deletion

SSM4 was isolated as a suppressor ofrna14-1, a mutant involved in nuclear mRNA maturation. In order to isolate genes interacting withSSM4, we have searched for mutants that are syntheticlethal in association with anSSM4 deletion. Among the mutants obtained, one, namedsls1-1, shows apet− phenotype. We have cloned and sequenced this gene. It encodes a protein with a calculated molecular mass of 73 kDa. This protein contains a mitochondrial targeting presequence but does not show homology with other known proteins. Deletion ofSLS1 does not affect cell viability on glucose but is lethal on a non-fermentable medium. The Sls1p protein does not appear to be involved in mitochondrial DNA replication, transcription, or in RNA splicing maturation or stability. We have also tagged this protein and localized it in mitochondria. Treatment with alkaline carbonate does not extract this protein from mitochondria, suggesting strongly that it is a mitochondrial integral membrane protein. Thus, theSLS1 gene, encodes a mitochondrial integral membrane protein and is paradoxically synlethal in association with a deletion of theSSM4 gene, which encodes an integral nuclear membrane protein.

Cytoplasmic CstF-77 protein belongs to a masking complex with cytoplasmic polyadenylation element-binding protein in Xenopus oocytes.

Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G2/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.

Effects of mutations in the Saccharomyces cerevisiae RNA14 gene on the abundance and polyadenylation of its transcripts

Abstract In the yeast Saccharomyces cerevisiae, the RNA14 and RNA15 gene products have been implicated in RNA cleavage and polyadenylation in vitro and in the choice of polyadenylation site of ACT1 mRNA in vivo. The RNA14 gene produces three transcripts that differ in their 3′ end, suggesting the use of different polyadenylation sites. The appearance of the three RNA14 transcripts was examined in different rna14 and/or rna15 mutant strains. In the rna14-1 or rna15-2 mutant strains, only the large transcript is present at the non-permissive temperature, showing that the rna14-1 and rna15-2 mutations lead to the use of the most distal RNA14 polyadenylation site, which turns out to be the most efficient. The rna14-5 mutation, which does not primarily modify the choice of poly(A) site, increases the global amount of RNA14 transcript. Surprisingly, this RNA14 mRNA overproduction is also observed in the double rna14-1 rna15-2 mutant strain. Moreover, in the strains in which the RNA14 transcripts are overproduced, short heterogeneous polyadenylated antisense RNAs are detected in the 3′ region of the RNA14 large transcript. Taken together these observations suggest that, in addition to poly(A) site choice, Rna14 protein has another function involved in the control of global RNA14 mRNA level.

Production of polyclonal antibodies against the S and preS2 regions of woodchuck hepatitis virus: lack of detectable low glycosylated preS2 protein (GP33) in sera from infected animals

Polyclonal antibodies directed against the preS2 and S domains of the woodchuck hepatitis virus (WHV) envelope proteins were prepared using synthetic peptides and fusion polypeptides as immunogens. They were tested by immunoblotting and immunoprecipitation of infected woodchuck sera and lysates of a eukaryotic cell line expressing WHV envelope proteins. Only one anti-peptide serum directed against the preS2 domain was reactive with WHV envelope proteins, recognizing the preS2 and preS1 proteins by their preS2 epitopes. With recombinant fusion proteins we generated several anti-S sera, which recognized all envelope proteins, and anti-preS2 antisera, which recognized the preS proteins. Results obtained with our antisera showed that sera of infected woodchucks lack the low glycosylated form (GP33) of the preS2 protein, unlike human hepatitis B virus.

Woodchuck hepatitis virus surface antigen produced in vitro fails to bind polymerized woodchuck serum albumin

Using a plasmid (pSWS) similar to one that has been successfully used for large-scale production of hepatitis B virus (HBV) envelope protein particles (pSVS) but containing the corresponding woodchuck hepatitis virus (WHV) envelope gene sequences, we have stably transformed the rodent dihydrofolate reductase-deficient cell line CHO dhfr-. Although production of WHV envelope particles in CHO/pSWS cell lines was low, it was sufficient to test whether these particles could bind to polymerized serum albumin. Whereas binding of HBV particles produced in CHO/pSVS cells to polymerized human serum albumin could readily be detected, we found no evidence that the WHV envelope protein particles produced in vitro bind to either human or woodchuck polymerized serum albumin.