Impact of nucleosome dynamics and histone modifications on cell proliferation during Arabidopsis development.

Eukaryotic chromatin is a highly structured macromolecular complex of which DNA is wrapped around a histone-containing core. DNA can be methylated at specific C residues and each histone molecule can be covalently modified at a large variety of amino acids in both their tail and core domains. Furthermore, nucleosomes are not static entities and both their position and histone composition can also vary. As a consequence, chromatin behaves as a highly dynamic cellular component with a large combinatorial complexity beyond DNA sequence that conforms the epigenetic landscape. This has key consequences on various developmental processes such as root and flower development, gametophyte and embryo formation and response to the environment, among others. Recent evidence indicate that posttranslational modifications of histones also affect cell cycle progression and processes depending on a correct balance of proliferating cell populations, which in the context of a developing organisms includes cell cycle, stem cell dynamics and the exit from the cell cycle to endoreplication and cell differentiation. The impact of epigenetic modifications on these processes will be reviewed here.

RNA: protein interactions associated with satellites of panicum mosaic virus.

The interactions between satellite panicum mosaic virus (SPMV) capsid protein (CP) and its 824 nucleotide (nt) single stranded RNA were investigated by gel mobility shift assay and Northwestern blot assay. SPMV CP has specificity for its RNA at high affinity, but little affinity for non-viral RNA. The SPMV CP also bound a 350 nt satellite RNA (satRNA) that, like SPMV, is dependent on panicum mosaic virus for its replication. SPMV CP has the novel property of encapsidating SPMV RNA and satRNA. Competition gel mobility shift assays performed with a non-viral RNA and unlabeled SPMV RNA and satRNA revealed that these RNA:protein interactions were in part sequence specific.

Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines.

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC(50) of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID(50) of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.

Genetic dissection of tomato bushy stunt virus p19-protein-mediated host-dependent symptom induction and systemic invasion.

The plus-sense single-stranded RNA of tomato bushy stunt virus (TBSV) encodes a 19-kDa protein, which is translated from a 3' proximal open reading frame (p19) that is entirely nested within the cell-to-cell movement gene (p22). Expression of the cytosolic p19-protein induces either a systemic lethal collapse in Nicotiana benthamiana and N. clevelandii, or necrotic local lesions on resistant N. tabacum. In spinach, the p19-protein is required at high abundance for efficient systemic invasion. This study aimed to determine whether these seemingly different host-dependent biological activities are governed by the same or separate regions on the 172 amino acid p19-protein. For this purpose, codons for charged amino acids predicted to be exposed on the surface of the polypeptide and presumably available for host-specific interactions, were targeted for mutagenesis. A total of 12 mutants were generated, which had no deficiencies in replication or cell-to-cell movement, and substitution of amino acids at the extreme N-terminal end or within the carboxyl 70 amino acids failed to cause a noticeable biological effect on plants. However, mutations dispersed between positions 43 and 85 on the N-terminal half prevented the onset of a systemic lethal necrosis on N. benthamiana and N. clevelandii. With one exception, the same mutants elicited mostly chlorotic, rather than necrotic, local lesions on N. tabacum. Mutations in the central region, which substituted Arg with Gly at positions 72 or 75-78, impaired the ability of TBSV to systemically invade spinach plants. However, substitution with Ala instead of Gly at position 72 had minimal effects on systemic spread in spinach, suggesting the possible influence of protein structure effects. The implications are that regions on the N-terminal portion of the p19-protein mediate interactions in a host-dependent manner and that a central region is required for all activities either by a direct effect of the amino acids or through maintenance of structural integrity.

A gene cluster encoded by panicum mosaic virus is associated with virus movement.

A subgenomic RNA (sgRNA) of about 1500 nucleotides has been detected in millet plants and protoplasts infected with panicum mosaic virus (PMV). This sgRNA expressed p8, p6.6, p15, and the 26-kDa capsid protein (CP) genes during in vitro translation assays, as determined by using mutants inactivated for expression of each open reading frame. Abolishing expression of p8 and p6.6, the two 5'-proximal genes on the sgRNA, did not affect the replication of PMV in millet protoplasts, but obstructed spread in plants. As predicted for a typical cell-to-cell movement protein, p8 localized to the cell wall fraction of PMV-infected millet plants. The introduction of premature stop codons downstream of the PMV p15 start codon (p15*) abolished infectivity in planta, but did not impair replication in protoplasts. However, a delayed systemic infection in millet plants was supported by the p15aug(-) start codon mutant, which may reflect very low levels of expression from a suboptimal start codon context and/or leaky scanning to a second inframe AUG codon to express the C-terminal portion of the 15-kDa protein. PMV CP mutants had little effect on sgRNA accumulation, but were correlated with a reduction of the gRNA and the decreased expression of the 8-kDa protein in protoplasts as well as abolishment of cell-to-cell spread in plants. These results imply that the successful establishment of a PMV systemic infection in millet host plants appears to be dependent on the concerted expression of the p8, p6.6, p15, and CP genes.

Identification of a biological inactive complex form of pokeweed antiviral protein.

Pokeweed antiviral protein (PAP) inactivates both eukaryotic and prokaryotic ribosomes via a specific depurination of rRNA. The sensitivity of pokeweed ribosomes to PAP implies the existence of a mechanism to protect the plant. Using monoclonal antibodies specific to PAP, a protein complex (PAPi) which contained PAP was identified in leaf extract. In this complex, the enzymatic activity of the toxin was strongly inhibited. This protein complex had a pI lower than that of PAP and was separated from free PAP by a preparative native gel electrophoresis. PAPi had an apparent molecular mass of 57 kDa and was dissociated by heating for 5 min at 80 degrees C or by treatment by alkaline or acidic pH or by 7 M urea. The other components involved in the complex remain unknown.

Production and characterization of monoclonal antibodies against the ribosome-inactivating protein PAP from Phytolacca americana.

Monoclonal antibodies specific to pokeweed antiviral protein (PAP), a ribosome-inactivating protein (RIP), were obtained after six unsuccessful fusions. A special procedure including injections of low doses of purified PAP from spring leaves in a short period was adopted. Some clones highly specific to PAP react with recombinant PAP. One clone cross-reacts with PAP-S isolated from seeds but none cross-reacts with the isoform PAP II isolated from summer leaves. These antibodies represent a useful tool to investigate the mechanisms of PAP biosynthesis and plant protection involving RIPs.