Identification of the mammalian Not gene via a phylogenomic approach.

Despite the great morphological diversity of early embryos, the underlying mechanisms of gastrulation are known to be broadly conserved in vertebrates. However, a number of genes characterized as fulfilling an essential function in this process in several model organisms display no clear ortholog in mammalian genomes. We have devised an in silico phylogenomic approach, based on exhaustive similarity searches in vertebrate genomes and subsequent bayesian phylogenetic analyses, to identify such missing genes, presumed to be highly divergent. This approach has been used to identify mammalian orthologs of Not, an homeodomain containing gene previously characterized in Xenopus, chick and zebrafish as playing a critical role in the formation of the notochord. This attempt led to the identification of a highly divergent mammalian Not-related gene in the mouse, human and rat. The results from phylogenetic reconstructions, synteny analyses, expression pattern analyses in wild-type and mutant mouse embryos, and overexpression experiments in Xenopus embryos converge to confirm these genes as representatives of the Not family in mammals. The identification of the mammalian Not gene delivers an important component for the understanding of the genetics underlying notochord formation in mammals and its evolution among vertebrates. The phylogenomic method used to retrieve this gene thus provides a tool, which can complement or validate genome annotations in situations when they are weakly supported.

European doctorate in biotechnology: Added value for european academia and industry.

It has long been recognized that educational programs and degrees are not equivalent across Europe. Add to this the fact that Europe consists of many different cultures and languages, then it is not surprising that the free circulation of scientists and their job market in the European Union is severely restricted. This is one of several debated causes for the crisis in European biotechnology, which is in danger of succumbing to the competition of North America, Japan, and some of the developing countries. The Universities (even those tradition-ridden), the European University Association, and the European Commission are aware of the danger and plans are in preparation for sweeping organizational and cultural changes. The problem is how long will it take and how long can we afford to wait? A number of biotechnologists and scientists from several institutions and many countries decided, instead of waiting, to make a preliminary move in the right direction. With the help of the European Commission and using European Molecular Biology Organization, European Federation of Biotechnology, and the European University Rectors as references, the European Association for Higher Education in Biotechnology was founded in 1995 by representatives of universities and research institutes. It awards the additional title of European Doctor to PhD graduates showing excellence in biotechnology and/or related Life Science subjects and who are willing to fulfil a program of studies that is both international and interdisciplinary. The present article reports on the first 9 years of this adventure.

In vivo functional analysis of ezrin during mouse blastocyst formation.

During mouse blastocyst formation, a layer of outer cells differentiates in less than 48 h into a functional epithelium (the trophectoderm). Ezrin, an actin-binding structural component of microvilli in epithelial cells, is also involved in signal transduction and ionic pump control. In the mouse embryo, ezrin becomes restricted to the apical cortex of all blastomeres at compaction and of outer cells at later stages. Here we investigated the function of ezrin in living embryos during epithelial differentiation using mutant forms of ezrin tagged with green fluorescent protein (GFP). GFP-tagged wild-type ezrin (Ez/GFPc) behaved like endogenous ezrin and did not interfere with development. Deletion of the last 53 amino acids (Delta53/GFP) changed the localization of ezrin: after compaction, Delta53/GFP remained associated with the apical and basolateral cortex in all blastomeres, and its expression slightly disturbed the cavitation process. Finally, full-length ezrin with GFP inserted at position 234 (Ez/GFPi) was localized all around the cortex throughout development, although it was concentrated at the apical pole after compaction. In embryos expressing Ez/GFPi, the duration of the 16-cell stage was reduced, while the onset of cavitation was delayed. Moreover, cavitation was abnormal, and the blastocoele was small and retracted almost completely several times as if there were major leakages of blastocoelic fluid. Our results suggest that, in addition to its role in microvilli organization, ezrin is involved in the formation of a functional epithelium through a still unknown mechanism.

A major posttranslational modification of ezrin takes place during epithelial differentiation in the early mouse embryo.

The preimplantation development of the mouse embryo leads to the formation of two populations of cells: the trophectoderm, which is a perfect epithelium, and the inner cell mass. The divergence between these two lineages is the result of asymmetric divisions, which can occur after blastomere polarization at compaction. The apical pole of microvilli is the only asymmetric feature maintained during mitosis and polarity is reestablished only in daughter cells that inherit all or a sufficient part of this pole. To analyze the role of ezrin in the formation and stabilization of the pole of microvilli, we isolated and cultured inner cell masses (ICM). These undifferentiated cells can differentiate very quickly into epithelial cells. After isolation of the ICMs, ezrin relocalizes at the cell cortex before the formation of microvilli. This redistribution occurs in the absence of protein synthesis. The formation of microvilli at the apical surface of the outer cells of ICM correlates with a major posttranslational modification of ezrin. We show here that this posttranslational modification is not controlled by a serine/threonine kinase but an O-glycosylation may partially contribute to it. These data suggest that ezrin has at least two roles during development. First, ezrin may be involved in the formation of microvilli because it localizes at the cell cortex before microvilli appear in ICMs. Second, ezrin may stabilize the pole of microvilli because it is modified posttranslationally when microvilli form.

Ezrin becomes restricted to outer cells following asymmetrical division in the preimplantation mouse embryo.

During preimplantation development in the mouse, two phenotypically distinct cell populations appear at the 16-cell stage: nonpolarized inner cells that give rise to the inner cell mass and polarized outer cells that give rise mainly to the trophectoderm. The divergence of these two cell lineages is due to asymmetrical cell divisions during the transition from the 8- to the 16 cell stage which can occur following blastomere polarization. During compaction, at the 8-cell stage, cytoplasmic organelles accumulate in the apical domain, a surface pole of microvilli forms, and blastomeres flatten onto one another. During the division from the 8- to the 16-cell stage, the only asymmetrical structure maintained is the pole of microvilli. At the 16-cell stage, only blastomeres inheriting a large part of this apical structure can reestablish a polarized organization. The mechanisms involved in the formation and stabilization of the apical pole of microvilli are still unknown. Ezrin is an actin-associated protein that has been proposed to play a role in the formation of microvillous structures. This led us to study the expression of ezrin during early development of the mouse embryo. We observed that ezrin mRNA and protein are present in the mouse oocyte and throughout preimplantation embryo development, although the amount of protein present decreases continuously during early development, particularly after the 8-cell stage, at the time of compaction. Two isoforms of ezrin phosphorylated on tyrosine residues are present during all of preimplantation development while a third non-tyrosine-phosphorylated isoform appears at the 8-cell stage and its relative amount increases from the 8-cell stage to the blastocyst stage. Before compaction, ezrin is distributed around the cell cortex. However ezrin becomes restricted to the microvilli of the apical pole after compaction. At later stages, ezrin is found in the microvilli of the apical surface of outer cells. Finally, ezrin remains associated with the microvillous pole during the transition from the 8- to 16-cell stage and is found only in the outer cells after division. Thus, ezrin is the first cytocortical protein described that is totally segregated in outer cells at the 16-cell stage after an asymmetrical division.

Evidence of singlet oxygen evolution by whole living cells of Chlamydomonas reinhardtii.

The oxygen evolved by Chlamydomonas reinhardtii in the light is measured simultaneously with a Clark electrode and with the nitrosodimethylaniline-imidazole colorimetric method which is specific for singlet oxygen. Experiments with wild-type and FuD7 mutant cells (unable to synthesize the D1 protein of Photosystem II), with dichlorophenyldimethylurea (which blocks electron transfer from Photosystem II to Photosystem I) and with dibromothymoquinone (which diverts electrons from their normal path between the two photosystems), as well as with hydroxylamine (an inactivator of the water-splitting part of Photosystem II and a competitor of water for electron donation to it), all point to the dependence of detected singlet oxygen on photolysis of water by Photosystem II.

Cell adhesion and gap junction formation in the early mouse embryo are induced prematurely by 6-DMAP in the absence of E-cadherin phosphorylation.

Compaction of the mouse embryo, which takes place at the 8-cell stage, is dependent upon the adhesion molecule E-cadherin (uvomurulin), but does not require protein synthesis, suggesting that post-translational modification(s) is (are) implicated in the setting up of this phenomenon. The demonstration recently that E-cadherin is phosphorylated at the 8-cell stage just before compaction supports this theory. In this work we used 6-dimethylaminopurine, a serine-threonine kinase inhibitor, to investigate the role of protein phosphorylation in compaction of mouse embryos. 6-dimethylaminopurine is able to induce cell flattening and gap junction formation prematurely at the 4-cell stage; however, it does not induce cell surface polarization, as occurs during normal compaction. 6-dimethylaminopurine-induced premature flattening is inhibited when the embryos are cultured in the presence of an anti-E-cadherin antibody or without extra-cellular Ca2+, demonstrating that this process requires functional E-cadherin; whereas cell flattening and gap junction formation take place in the absence of E-cadherin phosphorylation, suggesting that its phosphorylation is not required normally for these events. The relationship between E-cadherin-mediated cell flattening and gap junction formation during compaction is discussed.

gamma-Tubulin is present in acentriolar MTOCs during early mouse development.

gamma-Tubulin, a recently discovered member of the tubulin superfamily, is a peri-centriolar component considered to be essential for microtubule nucleation. Mouse oocytes and early embryos lack centrioles until the blastocyst stage. Thus, early mouse embryos allowed us to study the location of gamma-tubulin in animal cells in the absence of centrioles. For this, we used an antiserum directed against a specific peptide of the gamma-tubulin sequence, which is conserved among species. This serum has been characterised both in PtK2 and mouse cells. We found that it specifically-stained the spindle poles and the cytoplasmic microtubule organizing centers in metaphase II oocytes and the spindle poles in mitosis during the cleavage stages. In contrast, no interphase staining could be detected during cleavage. Since the overall level of gamma-tubulin did not decrease during interphase, as shown by immunoblotting experiments, this absence of staining during interphase is probably due to a cytoplasmic dispersion of gamma-tubulin. A single dot-like interphase reactivity appeared at the 32-cell stage. In parallel, electron microscopy studies allowed us to detect centrioles for the first time at the 64-cell stage. The possible roles of gamma-tubulin in microtubule nucleation and in centrosome maturation are discussed.

Bacteriorhodopsin "detergent-monomers," blue shift and velocity of light-dark adaptation.

Bacteriorhodopsin was incubated in various detergent solutions. Absorbance, circular dichroism and size of fragments obtained were investigated. Among all the detergents used, only Triton-X-100 and Nonidet P 40 led to monomers of bacteriorhodopsin. The blue shift of the absorbance maximum of the dark-adapted form and the slow down of dark adaptation are the sole parameters affected by the disruption of the trimeric organization of bacteriorhodopsin. Other spectral characteristics, such as reduction of amplitude of light adaptation, are affected by the presence of detergents independently of the associated or dispersed state of the pigment.

Complex formation between chlorophyll a and cytochrome c: surface properties at the air-water interface. Absorbance, fluorescence and fluorescence-lifetime in Langmuir-Blodgett films.

The binding of cytochrome c to an insoluble monolayer of chlorophyll a was studied. Surface pressure (II), surface potential (delta V) and [14C]cytochrome c surface-concentration (gamma) isotherms were measured versus molecular area (sigma) in mixed films. Compared to the successive-addition method, this procedure allows the formation of homogeneous mixed films. The cytochrome c is incorporated into a chlorophyll a monolayer, compressed at a surface pressure of 20 mN.m-1. On expansion, the quantity of protein incorporated into the monolayer gradually increases. Subsequent compression-expansion cycles result in similar isotherms, distinct from that measured during the first expansion. All surface properties measured, but more specifically the surface radioactivity of [14C]cytochrome c, indicate the irreversibility of protein incorporation into the chlorophyll a monolayer. In fact, surface properties of the binary film are completely different from the properties of either of the pure components. As a result, calculated values of surface potentials for mixed films using the additivity law deviate from experimentally measured potentials. The absorption and fluorescence spectra of mixed films transferred onto a solid substrate by the Langmuir-Blodgett technique, indicate a dilution effect of chlorophyll a by cytochrome c. However, the dilution effect cannot be detected by the fluorescence lifetimes of pure chlorophyll a and mixed chlorophyll a-cytochrome c films, both shorter than 0.2 ns. This provides support for the existence of an energy-transfer mechanism between chlorophyll a monomer and chlorophyll a aggregates which could serve as an energy trap. The role of the protein could be related to that of the matrix.

Cell polarity and microtubule organisation during mouse early embryogenesis.

We have studied the distribution and the role of microtubules in the major developmental events occurring during early development of the mouse. These events are the setting up of asymmetries within blastomeres, the process of asymmetrical cell division and the changes in cellular organisation taking place during epithelial differentiation.

Cytoskeleton organization during oogenesis, fertilization and preimplantation development of the mouse.

The organization and role of the cytoskeletal networks (mainly microtubules and microfilaments) during oogenesis, fertilization and preimplantation development of the mouse are described given the importance of cell-cell interactions and of the subcellular organization in events leading to the formation of the first two lineages of the mouse embryo.

Molecular cloning and characterization of comC, a late competence gene of Bacillus subtilis.

comC is a Bacillus subtilis gene required for the development of genetic competence. We have cloned a fragment from the B. subtilis chromosome that carries comC and contains all the information required to complement a Tn917lac insertion in comC. Genetic tests further localized comC to a 2.0-kilobase HindIII fragment. Northern (RNA) blotting experiments revealed that an 800-base-pair comC-specific transcript appeared at the time of transition from exponential to stationary phase during growth through the competence regimen. The DNA sequence of the comC region revealed two open reading frames (ORFs), transcribed in the same direction. The upstream ORF encoded a protein with apparent sequence similarity to the folC gene of Escherichia coli. Insertion of a chloramphenicol resistance determinant into this ORF and integration of the disrupted construct into the bacterial chromosome by replacement did not result in competence deficiency. The downstream ORF, which contained the Tn917lac insertion that resulted in a lack of competence, is therefore the comC gene. The predicted protein product of comC consisted of 248 amino acid residues and was quite hydrophobic. The comC gene product was not required for the expression of any other com genes tested, and this fact, together with the marked hydrophobicity of ComC, suggests that it may be a component of the DNA-processing apparatus of competent cells.

Light adaptation of bacteriorhodopsin in the presence of valinomycin and potassium. pH-dependence.

In the presence of valinomycin and K(+), bacteriorhodopsin undergoes (i) a decrease of its maximum absorbance, (ii) a blue shift of the maximum wavelength of both the light and the dark adapted forms. However (iii) a normal light adaptation is maintained and (iv) the retinal-retinal interactions are not perturbed. The role of valinomycin as a K(+)-carrier allowing a H(+)-K(+) competition as well as the stabilization of the deprotonated Schiff-base (linking retinal to the apo-opsin) is shown and discussed.

T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). Our results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

Purification of cardiolipin for surface pressure studies.

Thin-layer chromatography and surface pressure-area isotherms of commercial bovine cardiolipins showed that the samples contained contaminants. They were purified by TLC and their purity was checked by chromatography and by their monolayer properties. The molecular area of cardiolipin and its purification yield depend upon the fatty acid composition, particularly the degree of unsaturation.

Interactions of CF1, the coupling factor of photophosphorylations, with chloroplast lipids. Monolayers studies.

Surface pressure and surface potential of monolayers at a water-air interface show no polar interactions between the chloroplast total lipids and the polar protein CF1. The two components are perfectly miscible in mixed monolayers. It follows that CF1 interacts with the hydrophobic parts of the lipid molecules and that, in vivo, CF1 could penetrate in the hydrophobic core of the membranes.

Temperature dependent expression of polyoma virus in murine embryonal carcinoma cells.

At 37 degrees C, undifferentiated murine teratocarcinoma cells (PCC4) are resistant to infection with SV40 and Polyoma virus. When infection is carried out at 31 degrees C, these cells become fully susceptible to a variety of polyoma virus strains, including wt, ts-a, and hr-t; they also display an increased susceptibility to polyoma virus mutants (PyE.C.) which have been selected for their ability to develop in PCC4 cells at 37 degrees C (Vasseur et al., '80). However, expression of SV40 is still restricted at 31 degrees C and no T antigen can be detected. PCC4 cells grown at 31 degrees C express the characteristic embryonal surface antigen(s), but no H2 antigen, and do not produce plasminogen activator. PyE.C. mutants and other polyoma virus strains cannot develop at 37 degrees C in nullipotent F9 embryonal carcinoma cells and restriction is not abolished at 31 degrees C. The results indicate that: i) Resistance of PCC4 cells to polyoma virus and to SV40 are not mediated by the same process; ii) loss of restriction of polyoma in PCC4 cells does not require cell differentiation; iii) F9 and PCC4 cells control polyoma virus expression through different mechanisms.