Chfr interacts and colocalizes with TCTP to the mitotic spindle.

Chfr is a checkpoint protein that plays an important function in cell cycle progression and tumor suppression, although its exact role and regulation are unclear. Previous studies have utilized overexpression of Chfr to determine the signaling pathway of this protein in vivo. In this study, we demonstrate, by using three different antibodies against Chfr, that the endogenous and highly overexpressed ectopic Chfr protein is localized and regulated differently in cells. Endogenous and lowly expressed ectopic Chfr are cytoplasmic and localize to the spindle during mitosis. Higher expression of ectopic Chfr correlates with a shift in the localization of this protein to the nucleus/PML bodies, and with a block of cell proliferation. In addition, endogenous and lowly expressed ectopic Chfr is stable throughout the cell cycle, whereas when highly expressed, ectopic Chfr is actively degraded during S-G2/M phases in an autoubiquitination and proteasome-dependent manner. A two-hybrid screen identified TCTP as a possible Chfr-interacting partner. Biochemical analysis with the endogenous proteins confirmed this interaction and identified beta-tubulin as an additional partner for Chfr, supporting the mitotic spindle localization of Chfr. The Chfr-TCTP interaction was stable throughout the cell cycle, but it could be diminished by the complete depolymerization of the microtubules, providing a possible mechanism where Chfr could be the sensor that detects microtubule disruption and then activates the prophase checkpoint.

SNCF, a SoxNeuro interacting protein, defines a novel protein family in Drosophila melanogaster.

The involvement of the Sox family of transcription factors in the development of the central nervous system (CNS) appears to be conserved in invertebrates and vertebrates. In Drosophila, SoxNeuro (SoxN) was recently shown to be involved in the formation of neuroblasts [Development 129 (2002) 4193; Development 129 (2002) 4219]. Through a yeast two-hybrid assay searching for proteins interacting with SoxN, we have isolated a novel protein in Drosophila, SoxNeuro Co-Factor (SNCF). The expression of the SNCF gene was detected during early embryogenesis at the blastoderm stages, and stopped just at the beginning of gastrulation. In transfected cells, the protein localised to nuclei, and strongly accumulated in nucleoli. SNCF was able to enhance SoxN mediated transcriptional activity in transfected cells, suggesting that SNCF might act as a SoxN co-activator. Finally, data are presented showing the existence in Drosophila of several proteins with a domain of homology to SNCF, which are all expressed early in embryogenesis at the blastoderm stage.

SOX7 transcription factor: sequence, chromosomal localisation, expression, transactivation and interference with Wnt signalling.

The Sox gene family consists of several genes related by encoding a 79 amino acid DNA-binding domain known as the HMG box. This box shares strong sequence similarity to that of the testis determining protein SRY. SOX proteins are transcription factors having critical roles in the regulation of diverse developmental processes in the animal kingdom. We have characterised the human SOX7 gene and compared it to its mouse orthologue. Chromosomal mapping analyses localised mouse Sox7 on band D of mouse chromosome 14, and assigned human SOX7 in a region of shared synteny on human chromosome 8 (8p22). A detailed expression analysis was performed in both species. Sox7 mRNA was detected during embryonic development in many tissues, most abundantly in brain, heart, lung, kidney, prostate, colon and spleen, suggesting a role in their respective differentiation and development. In addition, mouse Sox7 expression was shown to parallel mouse Sox18 mRNA localisation in diverse situations. Our studies also demonstrate the presence of a functional transactivation domain in SOX7 protein C-terminus, as well as the ability of SOX7 protein to significantly reduce Wnt/beta-catenin-stimulated transcription. In view of these and other findings, we suggest different modes of action for SOX7 inside the cell including repression of Wnt signalling.

[Sex determination in mammals: state of the art]. / Détermination du sexe chez les mammifères: état des lieux.

Sex determination relies on the translation of chromosomal sex established at fertilisation into gonadal sex (testis or ovary), and later into somatic sex (male or female) under the control of gonadal hormone secretions. The aim of the current review will be to highlight our knowledge of the key events which, in the presence of a Y chromosome, induce the organisation of the developing epithelial cells located inside the genital ridges into testicular cords. Many groups have tried to define the molecules relevant to this process, with a double goal: unravelling a molecular pathway which leads to cell fate decision (Sertoli cell in this particular case) during development; improving the establishment of a diagnosis and subsequent medical management in cases where chromosomal, gonadal and then somatic sexes are discordant. Recent progress made in this area will be depicted, with the introduction of several pieces to this developmental jigsaw puzzle.

Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene.

For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

The human testis determining factor SRY binds a nuclear factor containing PDZ protein interaction domains.

The human Y-linked testis determining gene SRY encodes a protein with a DNA binding domain from the high mobility group box family. To date, no function has been assigned to amino acid sequences located outside this DNA binding motif. Here, we identify in a yeast two-hybrid screen a PDZ protein termed SIP-1, as an interacting protein with human SRY. In vitro, biochemical analysis, immunoprecipitation experiments, as well as expression of SIP-1 in human embryonic testis confirm that the two proteins can interact together. Interacting domains were mapped to the C-terminal seven amino acids of SRY and to the PDZ domains of SIP-1, respectively. We hypothesize that SIP-1 could connect SRY to other transcription factors providing SRY for its missing trans-regulation domain.

Mutations in STS1 suppress the defect in 3' mRNA processing caused by the rna15-2 mutation in Saccharomyces cerevisiae.

In a search for proteins associated with Rna15p in processing the 3' ends of messenger RNAs, we have looked for suppressors that correct, even partially, the thermosensitive growth defect of the rna15-2 mutant. Mutations in a single locus that we named SSM5, were able to suppress both the thermosensitivity of cell growth and the mRNA 3' processing defect associated with the rna15-2 mutation, but only slightly alleviated the thermosensitive growth defect of an rna14-1 mutant. The ssm5-1 mutant is sensitive to hydroxyurea at 37 degrees C, a drug that inhibits DNA synthesis. By screening for complementation of the hydroxyurea-sensitive phenotype we cloned the corresponding wild-type gene and found that it corresponds to the essential gene STS1 (also named DBF8). Sts1p has an apparent molecular weight of 30 kDa and was confirmed to be a cytosolic protein by immunofluorescence analysis. Western blot analysis indicates that the thermosensitive mutant strains rna15-2, rna14-1 and pap1-1 present a very low level of the Rna15p at 37 degrees C. The ssm5-1 mutation restores the level of Rna15p in the rna15-2 ssm5-1 double mutant. Use of the two-hybrid system suggests that Sts1p does not interact directly with Rna15p, but may be active as a homodimer. The present data suggest that Sts1p may play a role in the transport of Rna15p from the cytoplasm to the nucleus.

The duplicated Saccharomyces cerevisiae gene SSM1 encodes a eucaryotic homolog of the eubacterial and archaebacterial L1 ribosomal proteins.

A previously unknown Saccharomyces cerevisiae gene, SSM1a, was isolated by screening for high-copy-number suppressors of thermosensitive mutations in the RNA14 gene, which encodes a component from the polyadenylation complex. The SSM1 a gene codes for a 217-amino-acid protein, Ssm1p, which is significantly homologous to eubacterial and archaebacterial ribosomal proteins of the L1 family. Comparison of the Ssm1p amino acid sequence with that of eucaryotic polypeptides with unknown functions reveals that Ssm1p is the prototype of a new eucaryotic protein family. Biochemical analysis shows that Ssm1p is a structural protein that forms part of the largest 60S ribosomal subunit, which does not exist in a pool of free proteins. SSM1 a is duplicated. The second gene copy, SSM1b, is functional and codes for an identical and functionally interchangeable Ssm1p protein. In wild-type cells, SSM1b transcripts accumulate to twice the level of SSM1a transcripts, suggesting that SSM1b is responsible for the majority of the Ssm1p pool. Haploid cells lacking both SSM1 genes are inviable, demonstrating that, in contrast with its Escherichia coli homolog, Ssm1p is an essential ribosomal protein. Deletion of the most expressed SSM1b gene leads to a severe decrease in the level of SSM1 transcript, associated with a reduced growth rate. Polysome profile analysis suggests that the primary defect caused by the depletion in Ssm1p is at the level of translation initiation.

Cellular localization of RNA14p and RNA15p, two yeast proteins involved in mRNA stability.

RNA14 and RNA15 were originally identified by temperature-sensitive mutations that cause a rapid decrease in poly(A)-tail length and overall mRNA levels at the restrictive temperature. We have raised antibodies to the RNA14 and RNA15 proteins, and used subcellular fractionation and immunofluorescence to localize these proteins within the yeast cell. RNA14p is a 73 kDa protein found in both the nucleus and the cytoplasm, whilst RNA15p is a 42 kDa protein detected only in the nucleus. The observation that both proteins are found in the nucleus is in agreement with previous genetic data which suggest an interaction between RNA14p and RNA15p. Also the joint nuclear localization is consistent with the biochemical data suggesting a role in polyadenylation. The detection of significant amounts of RNA14p in the cytoplasm opens the possibility of a second function for this protein, either in cytoplasmic regulation of mRNA deadenylation or, more interestingly, in mRNA stability.

Cloning and expression in yeast of a plant potassium ion transport system.

A membrane polypeptide involved in K+ transport in a higher plant was cloned by complementation of a yeast mutant defective in K+ uptake with a complementary DNA library from Arabidopsis thaliana. A 2.65-kilobase complementary DNA conferred ability to grow on media with K+ concentration in the micromolar range and to absorb K+ (or 86Rb+) at rates similar to those in wild-type yeast. The predicted amino acid sequence (838 amino acids) has three domains: a channel-forming region homologous to animal K+ channels, a cyclic nucleotide-binding site, and an ankyrin-like region.

A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors.

We describe a set of replicative, integrative and single-stranded shuttle vectors constructed from the pUC19 plasmid that we use routinely in our experiments. They bear a yeast selectable marker: URA3, TRP1 or LEU2. Replicative vectors carrying different yeast replication origins have been constructed in order to have plasmids based on the same construction with a high or low copy number per cell and with different mitotic stabilities. All the vectors are small in size, provide a high yield in Escherichia coli and efficiently transform Saccharomyces cerevisiae. These plasmids have many of the unique sites of the pUC19 multicloning region and many of them allow for the screening of plasmids with an insert by alpha-complementation. The nucleotide sequence of each of them is completely known.

Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein.

In Saccharomyces cerevisiae, temperature-sensitive mutations in the genes RNA14 and RNA15 correlate with a reduction of mRNA stability and poly(A) tail length. Although mRNA transcription is not abolished in these mutants, the transcripts are rapidly deadenylated as in a strain carrying an RNA polymerase B(II) temperature-sensitive mutation. This suggests that the primary defect could be in the control of the poly(A) status of the mRNAs and that the fast decay rate may be due to the loss of this control. By complementation of their temperature-sensitive phenotype, we have cloned the wild-type genes. They are essential for cell viability and are unique in the haploid genome. The RNA14 gene, located on chromosome H, is transcribed as three mRNAs, one major and two minor, which are 2.2, 1.5, and 1.1 kb in length. The RNA15 gene gives rise to a single 1.2-kb transcript and maps to chromosome XVI. Sequence analysis indicates that RNA14 encodes a 636-amino-acid protein with a calculated molecular weight of 75,295. No homology was found between RNA14 and RNA15 or between RNA14 and other proteins contained in data banks. The RNA15 DNA sequence predicts a protein of 296 amino acids with a molecular weight of 32,770. Sequence comparison reveals an N-terminal putative RNA-binding domain in the RNA15-encoded protein, followed by a glutamine and asparagine stretch similar to the opa sequences. Both RNA14 and RNA15 wild-type genes, when cloned on a multicopy plasmid, are able to suppress the temperature-sensitive phenotype of strains bearing either the rna14 or the rna15 mutation, suggesting that the encoded proteins could interact with each other.

Monoclonal antibodies to the major capsid protein of human papillomavirus type 1.

Two stable monoclonal hybridoma cell lines secreting type-specific antibodies against the human papillomavirus type 1 (HPV-1) were isolated. The monoclonal antibodies detected HPV-1 antigens in frozen sections of HPV-1-induced warts, using immunofluorescence or immunoperoxidase techniques, and they reacted with HPV-1 particles in an immunodiffusion test. The two monoclonal antibodies recognized the major structural viral polypeptide, with a molecular weight of 54 000, and a minor polypeptide, with molecular weight of 76 000, in both the dissociated viral particles and in the wart extracts.

Physical state and transcription of the cottontail rabbit papillomavirus genome in warts and transplantable VX2 and VX7 carcinomas of domestic rabbits.

The physical state and the transcription of the genome of cottontail rabbit papillomavirus (CRPV) in non-virus-producing warts and in the VX2 and VX7 transplantable carcinomas of domestic rabbits were compared. The CRPV DNA present in VX2 and VX7 carcinomas (10 to 20 and 100 to 200 genome equivalents per diploid cell, respectively) was found to be entirely integrated into the cellular DNA, most probably as head-to-tail tandem repeats, in contrast to warts, in which viral DNA (10 to 100 copies per diploid cell) was found only as free, mainly monomeric, molecules. In the VX7 tumor, ca. 50% of the viral DNA molecules were found to be longer than one genome length, indicating that viral DNA rearrangements had occurred. A major viral transcript of 1,250 bases was detected in warts and in VX2 and VX7 carcinomas. Complementary sequences were localized within the E region, the putative transforming region inferred from the nucleotide sequence of the CRPV genome (I. Giri, O. Danos, and M. Yaniv, manuscript in preparation). Analysis of heteroduplexes formed between single-stranded CRPV DNA and polyadenylated RNAs from the VX2 tumor showed that the 1,250-base RNA resulted from the splicing of the sequences corresponding to the open reading frame E6 to those corresponding to the 3' third of E2. A second viral transcript, measuring 2,000 bases, was detected in warts and, in lesser amounts than the 1,250-base species, in VX2 carcinoma, and a 2,100-base RNA was found in VX7 carcinoma. Complementary sequences to these messengers were localized to the same part of the genome as the 1,250-base species and to a contiguous fragment situated upstream. Heteroduplex analysis showed that the 2,000-base species from VX2 carcinoma resulted from the splicing of the sequences corresponding to E6 and E7 to those corresponding to the 3' third of E2. The sequences spliced out upon the maturation of the two messengers of VX2 carcinoma correspond to E1, the two-thirds of E2, and most of E4. Additional transcripts were found in VX7 carcinoma, a major 3,100-base species transcribed from the E region, and several minor species, measuring from 2,400 bases, which all hybridize with a subgenomic fragment contained in the L region encoding the viral capsid polypeptides. This could account for the antiviral antibodies found in animals bearing the VX7 carcinoma.