Molecular genetic approaches to the biology of the moss "Physcomitrella patens"

La mousse haplobiontique Physcomitrella patens est utilisée comme système génétique modèle pour l'étude du développement des plantes. Cependant, l'absence d'un protocole efficace de transformation a constitué jusqu'à présent un gros désavantage méthodologique pour le développement futur de ce système expérimental. Les résultats présentés dans le premier chapitre relatent la mise au point d'un protocole de transformation basé sur la technique de transfert direct de gènes dans des protoplastes par précipitation au PEG. Un essai d'expression transitoire de gènes a été mis au point. Ce protocole a été adapté afin de permettre l'introduction in vivo d'anticorps dans des protoplastes. Le protocole modifié permet d'introduire simultanément du DNA et des IgG dans les cellules, et nous avons démontré que ces anticorps peuvent inactiver spécifiquement le produit d'un gène co-introduit (GUS), ainsi que certaines protéines impliquées dans des processus cellulaires (tubuline). Cet essai, baptisé "essai transitoire d'immuno-inactivation in vivo", devrait être directement applicable à d'autres protoplastes végétaux, et permettre l'élaboration de nouvelles stratégies dans l'étude de processus cellulaires. Le second chapitre est consacré aux expériences de transformation de la mousse avec des gènes conférant une résistance à des antibiotiques. Nos résultats démontrent que l'intégration de gènes de résistance dans le génome de P. patens est possible, mais que cet événement est rare. Il s'agit là néanmoins de la première démonstration d'une transformation génétique réussie de cet organisme. L'introduction de gènes de résistance aux antibiotiques dans les protoplastes de P. patens génère à haute fréquence des clones résistants instables. Deux classes de clones instables ont été identifiés. La caractérisation phénotypique, génétique et moléculaire de ces clones suggère fortement que les séquences transformantes sont concaténées pour former des structures de haut poids moléculaire, et que ces structures sont efficacement répliquées et maintenues dans les cellules résistantes en tant qu'éléments génétiques extrachromosomaux. Ce type de transformation nous permet d'envisager des expériences permettant l'identification des séquences génomiques impliquées dans la replication de l'ADN de mousse. Plusieurs lignées transgéniques ont été retransformées avec des plasmides portant des séquences homologues aux séquences intégrées dans le génome, mais conférant une résistance à un autre antibiotique. Les résultats présentés dans le troisième chapitre montrent que les fréquences de transformation intégrative dans les lignées transgéniques sont 10 fois plus élevées que dans la lignée sauvage, et que cette augmentation est associée à une coségrégation des gènes de résistance dans la plupart des clones testés. Ces résultats génétiques indiquent que l'intégration de séquences d'ADN étranger dans le génome de P. patens a lieu en moyenne 10 fois plus fréquemment par recombinaison homologue que par intégration aléatoire. Ce rapport homologue/aléatoire est 10000 fois supérieur aux rapports obtenus avec d'autres plantes, et fournit l'outil indispensable à la réalisation d'expériences de génétique inverse dans cet organisme à haplophase dominante. THESIS SUMMARY The moss Physcomitrella patens is used as a model genetic system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. But further development of this model system was hampered by the lack of a protocol allowing the genetic transformation of this plant. We have developed a transformation protocol based on PEG-mediated direct gene transfer to protoplasts. Our data demonstrate that this procedure leads to the establishment of an efficient transient gene expression assay. A slightly modified protocol has been developed allowing the in vivo introduction of antibodies in moss protoplasts. Both DNA and IgGs can be loaded simultaneously, and specific antibodies can immunodeplete the product of an expression cassette (GUS) as well as proteins involved in cellular processes (tubulins). This assay, named transient in vivo immunodepletion assay, should be applicable to other plant protoplasts, and offers new approaches to study cellular processes. Transformations have been performed with bacterial plasmids carrying antibiotic resistance expression cassette. Our data demonstrate that integrative transformation occurs, but at low frequencies. This is the first demonstration of a successful genetic transformation of mosses. Resistant unstable colonies are recovered at high frequencies following transformation, and two different classes of unstable clones have been identified. Phenotypical, genetic and molecular characterisation of these clones strongly suggests that bacterial plasmids are concatenated to form high molecular arrays which are efficiently replicated and maintained as extrachromosomal elements in the resistant cells. Replicative transformation in P. patens should allow the design of experiments aimed at the identification of genomic sequences involved in moss DNA replication. Transgenic strains have been retransformed with bacterial plasmids carrying sequences homologous to the integrated transloci, but conferring resistance to another antibiotic. Our results demonstrate an order of magnitude increase of integrative transformation frequencies in transgenic strains as compared to wild-type, associated with cosegregation of the resistance genes in most of these double resistant transgenic strains. These observations provide strong genetic evidence that gene targeting occurs about ten times more often than random integration in the genome of P. patens. Such ratio of targeted to random integration is about 10 000 times higher than previous reports of gene targeting in plants, and provides the essential requirement for the development of efficient reverse genetics in the haplodiplobiontic P. patens.

Diffusionless first-order phase transitions in systems with frozen configurational degrees of freedom

We consider systems that can be described in terms of two kinds of degree of freedom. The corresponding ordering modes may, under certain conditions, be coupled to each other. We may thus assume that the primary ordering mode gives rise to a diffusionless first-order phase transition. The change of its thermodynamic properties as a function of the secondary-ordering-mode state is then analyzed. Two specific examples are discussed. First, we study a three-state Potts model in a binary system. Using mean-field techniques, we obtain the phase diagram and different properties of the system as a function of the distribution of atoms on the different lattice sites. In the second case, the properties of a displacive structural phase transition of martensitic type in a binary alloy are studied as a function of atomic order. Because of the directional character of the martensitic-transition mechanism, we find only a very weak dependence of the entropy on atomic order. Experimental results are found to be in quite good agreement with theoretical predictions.

Critical behavior of a system with orientational and positional degrees of freedom: A Monte Carlo simulation study

The critical behavior of a system constituted by molecules with a preferred symmetry axis is studied by means of a Monte Carlo simulation of a simplified two-dimensional model. The system exhibits two phase transitions, associated with the vanishing of the positional order of the center of mass of the molecules and with the orientational order of the symmetry axis. The evolution of the order parameters and the specific heat is also studied. The transition associated with the positional degrees of freedom is found to change from a second-order to a first-order behavior when the two phase transitions are close enough, due to the coupling with the orientational degrees of freedom. This fact is qualitatively compared with similar results found in pure liquid crystals and liquid-crystal mixtures.

Domain growth in binary mixtures at low temperatures

We have studied domain growth during spinodal decomposition at low temperatures. We have performed a numerical integration of the deterministic time-dependent Ginzburg-Landau equation with a variable, concentration-dependent diffusion coefficient. The form of the pair-correlation function and the structure function are independent of temperature but the dynamics is slower at low temperature. A crossover between interfacial diffusion and bulk diffusion mechanisms is observed in the behavior of the characteristic domain size. This effect is explained theoretically in terms of an equation of motion for the interface.

Triplet pairing in fermionic droplets

We have investigated, in the L-S coupling scheme, the appearance of triplet pairing in fermionic droplets in which a single nl shell is active. The method is applied to a constant-strength model, for which we discuss the different phase transitions that take place as the number of particles in the shell is varied. Drops of 3He atoms can be plausible physical scenarios for the realization of the model.

Thermal nucleation and cavitation in 3He and 4He

Density functionals that reproduce the helium liquid-gas interface as a function of temperature have been used, within an improved homogeneous nucleation approach, to investigate thermal nucleation and cavitation in both helium isotopes. The results are compared with available experimental data on cavitation in 3He and 4He. Predictions are made for cavitation in 3He at negative pressures and for nucleation in both isotopes.

Effects of domain morphology in phase-separation dynamics at low temperature

We present numerical results of the deterministic Ginzburg-Landau equation with a concentration-dependent diffusion coefficient, for different values of the volume fraction phi of the minority component. The morphology of the domains affects the dynamics of phase separation. The effective growth exponents, but not the scaled functions, are found to be temperature dependent.

Stability of a nonequilibrium steady-state interface

We study the interfacial modes of a driven diffusive model under suitable nonequilibrium conditions leading to possible instability. The external field parallel to the interface, which sets up a steady-state parallel flux, enhances the growth or decay rates of the interfacial modes. More dramatically, asymmetry in the model can introduce an oscillatory component into the interfacial dispersion relation. In certain circumstances, the applied field behaves as a singular perturbation.

Fluctuations in domain growth: Ginzburg-Landau equations with multiplicative noise

Ginzburg-Landau equations with multiplicative noise are considered, to study the effects of fluctuations in domain growth. The equations are derived from a coarse-grained methodology and expressions for the resulting concentration-dependent diffusion coefficients are proposed. The multiplicative noise gives contributions to the Cahn-Hilliard linear-stability analysis. In particular, it introduces a delay in the domain-growth dynamics.

Numerical algorithm for Ginzburg-Landau equations with multiplicative noise: Application to domain growth

We consider stochastic partial differential equations with multiplicative noise. We derive an algorithm for the computer simulation of these equations. The algorithm is applied to study domain growth of a model with a conserved order parameter. The numerical results corroborate previous analytical predictions obtained by linear analysis.

Front and domain growth in the presence of gravity

Front and domain growth of a binary mixture in the presence of a gravitational field is studied. The interplay of bulk- and surface-diffusion mechanisms is analyzed. An equation for the evolution of interfaces is derived from a time-dependent Ginzburg-Landau equation with a concentration-dependent diffusion coefficient. Scaling arguments on this equation give the exponents of a power-law growth. Numerical integrations of the Ginzburg-Landau equation corroborate the theoretical analysis.

High-pressure ultrasonic study of vibrational anharmonicity in bcc Cu-Al-Be alloys

To quantify the vibrational anharmonicity of the long-wavelength acoustic modes of bcc Cu74.1Al23.1Be2.8 near its martensitic transition temperature Ms (261 K), the hydrostatic pressure derivatives (¿CIJ/¿P)P=0 of the elastic stiffness moduli have been measured. The Grüneisen parameters at 268 K (just above Ms), especially of longitudinal modes, which become smaller than those of the shear modes, are quite different from those at 295 K: the anharmonicity changes markedly in the vicinity of the transition. Similar trends are noted for Cu66.5Al12.7Zn20.8. Experimental data near Ms are used to estimate cubic invariants in the strain order parameters in a Landau formalism.