Functional organization of the cassava vein mosaic virus (CsVMV) promoter.

Cassava vein mosaic virus (CsVMV) is a pararetrovirus that infects cassava plants in Brazil. A promoter fragment isolated from CsVMV, comprising nucleotides -443 to +72, was previously shown to direct strong constitutive gene expression in transgenic plants. Here we report the functional architecture of the CsVMV promoter fragment. A series of promoter deletion mutants were fused to the coding sequence of uidA reporter gene and the chimeric genes were introduced into transgenic tobacco plants. Promoter activity was monitored by histochemical and quantitative assays of beta-glucuronidase activity (GUS). We found that the promoter fragment is made up of different regions that confer distinct tissue-specific expression of the gene. The region encompassing nucleotides -222 to -173 contains cis elements that control promoter expression in green tissues and root tips. Our results indicate that a consensus as1 element and a GATA motif located within this region are essential for promoter expression in those tissues. Expression from the CsVMV promoter in vascular elements is directed by the region encompassing nucleotides -178 to -63. Elements located between nucleotides -149 and -63 are also required to activate promoter expression in green tissues suggesting a combinatorial mode of regulation. Within the latter region, a 43 bp fragment extending from nucleotide -141 to -99 was shown to interact with a protein factor extracted from nuclei of tobacco seedlings. This fragment showed no sequence homology with other pararetrovirus promoters and hence may contain CsVMV-specific regulatory cis elements.

Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants.

The genes encoding the heavy and light chains of a murine monoclonal antibody (mAb Guy's 13) have been cloned and expressed in Nicotiana tabacum. Transgenic plants have been regenerated that secrete full-length Guy's 13 antibody. By manipulation of the heavy chain gene sequence, constant region domains from an immunoglobulin alpha heavy chain have been introduced, and plants secreting Guy's 13 mAb with chimeric gamma/alpha heavy chains have also been produced. For each plant antibody, light and heavy chains have been detected by Western blot analysis and the fidelity of assembly confirmed by demonstrating that the antibody is fully functional, by antigen binding studies. Furthermore, the plant antibodies retained the ability to aggregate streptococci, which confirms that the bivalent antigen-binding capacity of the full length antibodies is intact. The results demonstrate that IgA as well as IgG class antibodies can be assembled correctly in tobacco plants and suggest that transgenic plants may be suitable for high-level expression of more complex genetically engineered immunoglobulin molecules. Since mAb Guy's 13 prevents streptococcal colonization in humans, transgenic plant technology may have therapeutic applications.