Heterologous complementation of the exopolysaccharide synthesis and carbon utilization phenotypes of Sinorhizobium meliloti Rm1021 polyhydroxyalkanoate synthesis mutants

A reduced exopolysaccharide phenotype is associated with inability to synthesize polyhydroxyalkanaote (PHA) stores in Sinorhizobium meliloti strain Rm1021. Loss of function mutations in phbB and phbC result in non-mucoid colony morphology on Yeast Mannitol Agar, compared to the mucoid phenotype exhibited by the parental strain. This phenotype is attributed to reduction in succinoglycan synthesis. We have used complementation of this phenotype and the previously described D-3-hydroxybutyrate/acetoacetate utilization phenotype to isolate a heterologous clone containing a Bradyrhizobium japonicum phbC gene. Sequence analysis confirmed that this clone contains one of the five predicted phbC genes in the B. japonicum genome. The described phenotypic complementation strategy should be useful for isolation of novel PHA synthesis genes of diverse origin.     

Extensive phenotypic variation in early flowering mutants of Arabidopsis

Flowering time, the major regulatory transition of plant sequential development, is modulated by multiple endogenous and environmental factors. By phenotypic profiling of 80 early flowering mutants of Arabidopsis, we examine how mutational reduction of floral repression is associated with changes in phenotypic plasticity and stability. Flowering time measurements in mutants reveal deviations from the linear relationship between the number of leaves and number of days to bolting described for natural accessions and late flowering mutants. The deviations correspond to relative early bolting and relative late bolting phenotypes. Only a minority of mutants presents no detectable phenotypic variation. Mutants are characterized by a broad release of morphological pleiotropy under short days, with leaf characters being most variable. They also exhibit changes in phenotypic plasticity across environments for florigenic-related responses, including the reaction to light and dark, photoperiodic behavior, and Suc sensitivity. Morphological pleiotropy and plasticity modifications are differentially distributed among mutants, resulting in a large diversity of multiple phenotypic changes. The pleiotropic effects observed may indicate that floral repression defects are linked to global developmental perturbations. This first, to our knowledge, extensive characterization of phenotypic variation in early flowering mutants correlates with the reports that most factors recruited in floral repression at the molecular genetic level correspond to ubiquitous regulators. We discuss the importance of functional ubiquity for floral repression with respect to robustness and flexibility of network biological systems.     

Purification of a plant cell wall fibronectin-like adhesion protein involved in plant response to salt stress

The structural role of extracellular-matrix (ECM) has been recognized in both plants and animals as a support and anchorage-inducing cell behavior. Unlike the animal ECM proteins, the proteins that have been identified in plant ECM have not yet been purified from whole plants and cell wall. As several immunological data indicate the presence of animal ECM-like proteins in plants cell wall, especially under salt stress or water deficit, we propose a protocol to purify a fibronectin-like protein from the cell wall of epicotyls of young germinating peas. The process consists of a combination of gelatin and heparin affinity chromatography, close to the classical one used for human blood plasma fibronectin purification. Proteins with affinity for gelatin and heparin, immunologically related to human fibronectin, are found in the cell wall of epicotyls grown under salt stress or not. Total amount of purified proteins is 3-4 times more enriched in salt stressed epicotyls. SDS-PAGE and Western blot with antibodies directed against human blood plasma fibronectin give evidence that the cell wall proteins purified by gelatin/heparin affinity chromatography are closely related to human fibronectin. The present protocol leads us to purify 17 (control) or 65 (salt stress) micrograms of protein per g of fresh starting material. Our results suggest that plant cell wall proteins can provide better anchorage of the cell to its cell-wall during salt stress or water deficit and could be considered not only as cell adhesion but also as signaling molecules.     

Oncostatin M-stimulated apical plasma membrane biogenesis requires p27(Kip1)-regulated cell cycle dynamics

Oncostatin M regulates membrane traffic and stimulates apicalization of the cell surface in hepatoma cells in a protein kinase A-dependent manner. Here, we show that oncostatin M enhances the expression of the cyclin-dependent kinase (cdk)2 inhibitor p27(Kip1), which inhibits G(1)-S phase progression. Forced G(1)-S-phase transition effectively renders presynchronized cells insensitive to the apicalization-stimulating effect of oncostatin M. G(1)-S-phase transition prevents oncostatin M-mediated recruitment of protein kinase A to the centrosomal region and precludes the oncostatin M-mediated activation of a protein kinase A-dependent transport route to the apical surface, which exits the subapical compartment (SAC). This transport route has previously been shown to be crucial for apical plasma membrane biogenesis. Together, our data indicate that oncostatin M-stimulated apicalization of the cell surface is critically dependent on the ability of oncostatin M to control p27(Kip1)/cdk2-mediated G(1)-S-phase progression and suggest that the regulation of apical plasma membrane-directed traffic from SAC is coupled to centrosome-associated signaling pathways.     

Comparative vaccine studies in HLA-A2.1-transgenic mice reveal a clustered organization of epitopes presented in hepatitis C virus natural infection

A polyepitopic CD8(+)-T-cell response is thought to be critical for control of hepatitis C virus (HCV) infection. Using transgenic mice, we analyzed the immunogenicity and dominance of most known HLA-A2.1 epitopes presented during infection by using vaccines that carry the potential to enter clinical trials: peptides, DNA, and recombinant adenoviruses. The vaccines capacity to induce specific cytotoxic T lymphocytes and interferon gamma-producing cells revealed that immunogenic epitopes are clustered in specific antigens. For two key antigens, flanking regions were shown to greatly enhance the scope of epitope recognition, whereas a DNA-adenovirus prime-boost vaccination strategy augmented epitope immunogenicity, even that of subdominant ones. The present study reveals a clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.     

Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus

We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design.