Novel constructions to enable the integration of genes into the Agrobacterium tumefaciens C58 chromosome.

We constructed several versatile sets of vectors that can be used to introduce any gene into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome without affecting T-DNA transfer. One set contains a fragment containing the lacIq and lacZ genes and a multiple cloning site from pBluescriptII SK(+) inserted into a PstI site between the pgl and picA genes on an incPalpha plasmid. The resulting plasmid contains eight unique restriction endonuclease sites and the ability to use blue-white screening for the presence of an insert. A second plasmid also contains a beta-lactamase gene within this locus and provides a convenient ampicillin-carbenicillin resistance marker for the selection of genes integrated into the chromosome following double homologous recombination (homogenotization). A third plasmid contains, in addition to the lacZ, lacIq, and beta-lactamase genes within the pgl/picA locus, a sacRB gene cassette within the vector to counterselect against the presence of the vector within A. tumefaciens. To test this system, we introduced a wild-type virD2 gene into the A. tumefaciens chromosome at the pgl/picA locus. When a Ti plasmid harboring a deletion of virD2 was in this strain, the integrated virD2 gene complemented the virD2 deletion and the resulting transformation phenotype was identical to that resulting from A. tumefaciens strains harboring a wild-type virD2 gene located on a replicating plasmid.

Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus.

The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in transgenic potato plants under the control of a cauliflower mosaic virus 35S promoter or a modified 35S promoter that included two copies of a strong transcriptional enhancer. Both types of promoters allowed the production of specific mRNAs and detectable levels of recombinant VP60, which were higher for the constructs carrying the modified 35S promoter. Rabbits immunized with leaf extracts from plants carrying this modified 35S promoter showed high anti-VP60 antibody titers and were fully protected against the hemorrhagic disease.

Agrobacterium tumefaciens-mediated transformation of Pinus pinea L. cotyledons: an assessment of factors influencing the efficiency of uidA gene transfer.

This study is the first report of a protocol for transfer and expression of foreign chimeric genes into cotyledons excised from Pinus pinea L. embryos. Agrobacterium tumefaciens EHA105 harbouring the plasmid p35SGUSint was more infective than LBA4404 or C58 GV3850, as determined by the percentage of cotyledons showing uidA expression. Factors which significantly affected the T-DNA transfer included: (1) preinduction and concentration of bacteria, (2) days of coculture and (3) the wounding procedure applied. More efficient transfer of the uidA gene was achieved growing the bacteria in YEP medium at pH 7, infecting the cotyledons according to the sonication-assisted Agrobacterium-mediated transformation procedure with a bacterial density of 1 (OD600 nm) for 5 min, and coculture for 72 h. Using this protocol, 49.7% of the cotyledons showed a diffuse blue staining 7 days after infection. However, all were necrotic 30 days after inoculation. Since a decrease in bacterial density to 0.01 allowed the recovery of about 4% of cotyledons forming buds 1 month after inoculation, we conclude that the high mortality associated with the infection may be related to the hypersensitive response of the plant to bacterial infection.

Immunoreactivity for phosphorylated 200-kDa neurofilament subunit is heterogeneously expressed in human sympathetic and primary sensory neurons.

This study was undertaken to investigate whether human sensory and sympathetic neurons contain phosphorylated neurofilament proteins, and whether they may be classified on the basis of this property, as in other mammalian species. The distribution of the phosphorylated 200-kDa neurofilament protein subunit (p200-NFP) was investigated in lumbar sympathetic and dorsal root ganglia by means of the RT97 monoclonal antibody (against p200-NFP). The intensity of immunostaining, and the size of neuronal body profiles were measured in order to define different neuron subclasses. In dorsal root ganglia, most of the neuronal profiles (96%) were p200-NFP immunoreactive, and the intensity of immunostaining was not related to neuronal perikarya size. In the lumbar paravertebral sympathetic ganglia, virtually all neurons displayed p200-NFP immunoreactivity, and the intensity of immunolabelling was also independent of the size of the neuronal somata. These results demonstrate heterogeneity in the expression of p200-NFP immunoreactivity in human sympathetic and sensory neurons. In contrast to other mammalian species, RT97 immunolabelling cannot be used as a discriminative marker for the two main types of human primary sensory neurons. On the other hand, our findings provide evidence for the occurrence of phosphorylated neurofilaments within peripheral neuron cell bodies.