Sonchus yellow net rhabdovirus nuclear viroplasms contain polymerase-associated proteins.

We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of beta-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.

Fluorescent in situ hybridization of maize meiotic chromosomes as visualized by confocal microscopy.

Knob heterochromatin served as the model for the development of fluorescent chromosome in situ hybridization on maize meiotic chromosomes. The meiotic chromosomes were hybridized with a digoxigenin-labeled RNA probe of the knob repeat sequence that is a component of the morphologically determined knob heterochromatin. The fluorescein-labeled knob probe and propidium iodide counter-stained chromosomes were imaged using confocal laser scanning microscopy, which allowed for the individual analysis of each fluorescent probe emission intensity, the ability to utilize image processing techniques and the generation of high-resolution images. A composite, in register, merged image of the knob probe signal and meiotic chromosomes demonstrated exact co-localization of the knob probe and the morphologically identified knobs. The establishment of the fluorescent in situ hybridization technique in maize allows for the expanded study of the biological role of knob heterochromatin and the possibility of locating other repeat sequences on maize chromosomes.

The effect of chemical facilitators on the frequency of electrofusion of tobacco mesophyll protoplast.

Trypsin, pronase, protease, dispase, spermine, spermidine, and DMSO were characterized for their effect on the frequency of electrofusion of tobacco mesophyll protoplasts. Protease (Boehringer Mannheim) and Sigma pronase (1.26 mg/ml; 15 min incubation) increased the fusion frequency from 7% (control) to 20.7% (2.9X increase). Following protease and pronase were trypsin (2.8X), spermine (2.4X), dispase (2.1X), DMSO (2.0X), and spermidine (1.4X). BM Protease and polyamines caused the least amount of damage, followed by DMSO and trypsin (26% and 24% decrease in viability respectively), pronase (41%) and dispase (57%). Callus formed from all but dispase-treated protoplasts. Shoots regenerated from calli of all but trypsin-treated protoplasts.

Pyruvate orthophosphate dikinase: intracellular site of synthesis in maize leaf cells.

Pyruvate orthophosphate dikinase is synthesized in non-green leaf cells of the maize mutant iojap. Since iojap plastids lack ribosomes, it is concluded that the site of synthesis of pyruvate orthophosphate dikinase in maize leaf cells is on ribosomes in the cytoplasm.