Kathleen J. Danna

Accumulation of a thermostable endo-1,4-β-D-glucanase in the apoplast of Arabidopsis thaliana leaves.

Plant biomass, the most abundant renewable resource on earth, is a potential source of fermentable sugars for production of alternative transportation fuels and other chemicals. Bioconversion of plant biomass to fermentable glucose involves enzymatic hydrolysis of cellulose, a major polysaccharide constituent. Because commercially available microbial cellulases are prohibitively expensive for bioethanol processes, we have investigated the feasibility of producing these enzymes in plants as a low-cost, potentially high-volume alternative to traditional production methods. We have successfully expressed the catalytic domain of a thermostable (Topt=81xa0°C) endo-1,4-β-D-glucanase from the eubacterium, Acidothermus cellulolyticus, in the apoplast of tobacco BY-2 suspension cells and leaves of Arabidopsis thaliana plants. The apoplast-targeting cassette designed for this work consists of the cauliflower mosaic virus 35S promoter, the tobacco mosaic virus Ω translational enhancer, the sequence encoding the tobacco Pr1a signal peptide, and the polyadenylation signal of nopaline synthase. Recombinant E1 catalytic domain was targeted to the ER by the signal peptide and secreted into the apoplast via the default pathway. Secretion of the enzyme did not detectably affect the growth rate of transgenic BY-2 cells, although the protein was enzymatically active at elevated temperatures. Similarly, transgenic plants exhibited no abnormal phenotypes correlating with expression of the enzyme. Close agreement between independent immunochemical and activity-based assays indicates that the enzyme accumulated to concentrations up to 26% of the total soluble protein in leaves of primary A.xa0thaliana transformants. The amount of functional endoglucanase produced illustrates that plants can accumulate very large quantities of enzyme for commercial biomass conversion.

pBIN20: An Improved Binary Vector for shape Agrobacterium-mediated Transformation

We report the construction of a binary vector for Agrobacterium tumefaciens-mediated transformation, pBIN20, which contains a superlinker region located between the left and right Ti border sequences. This vector, derived from pBI121, simplifies the cloning of plant expression cassettes and has been used in our laboratory to create lines of transgenic BY-2 tobacco cells. This new vector contains more than 20 unique restriction sites as well as the nptII selectable marker gene within the Ti-DNA borders.

The amino-terminal 147 amino acids of SV40 large T antigen transform secondary rat embryo fibroblasts

T147D is an SV40 mutant that encodes only the amino-terminal 147 amino acids of large T antigen and does not make small t antigen. We have constructed a recombinant retrovirus that expresses the T147D mutant protein. We show here that this virus can transform the established rat cell line, F111, in an agar assay with high efficiency. More importantly, we demonstrate that this retrovirus transforms secondary rat embryo fibroblasts to anchorage independence as efficiently as a recombinant retrovirus that expresses both wild-type large and small T antigens. These data indicate that in rat cells, the amino-terminal 147 amino acids of T antigen are sufficient for transformation. Further, since the T147D protein does not bind p53, we conclude that the association between T antigen and p53 is not required for the transformation of rat cells to anchorage-independent growth.

A new SV40 mutant that encodes a small fragment of T antigen transforms established rat and mouse cells

We have constructed a new SV40 mutant, T147, that makes a large T antigen that is only 147 amino acids long. We show that the T147 T antigen is a phosphoprotein that is as stable as wild-type T antigen and that the SV40 viral origin binding activity of the T147 T antigen is reduced at least 100-fold relative to that of wild-type T antigen. Most importantly, we demonstrate that cloned T147 DNA transforms rat F111 cells to anchorage independence as efficiently as cloned wild-type SV40 DNA and that cloned T147 DNA also efficiently transforms C3H10T1/2 mouse cells in a focus assay.

An amino-terminal fragment of SV40 T antigen transforms REF52 cells

An SV40 mutant, T147D, encodes only the amino-terminal 147 amino acids of large T antigen and does not make small t antigen. We show here that a retrovirus which expresses this mutant T antigen transforms rat REF52 cells as efficiently as a retrovirus that expresses both the wild-type large and small T antigens. This cell line had previously been refractory to transformation by mutants that make short, amino-terminal fragments of T antigen.

An SV40 mutant oncoprotein has a nuclear location

T147 is an SV40 mutant that makes a normal small t antigen and a large T antigen that is only 147 amino acids long. We have introduced a second mutation into the genome of T147 which eliminates its ability to encode small t antigen. We show that this double mutant is able to transform C3H10T1/2 mouse cells in a focus assay and F111 rat cells in an agar suspension assay, demonstrating that the transforming domain of T antigen is located within its amino-terminal 147 amino acids. We also show that the T147 mutant T antigen, like wild-type T antigen, has a nuclear location. However, in contrast to wild-type T antigen, which is also found in the plasma membranes of wild-type transformed cells, we fail to detect any mutant T antigen associated with the plasma membranes of T147 transformants.

Efficient infection of monkey cells with SV40 DNA. II. Use of low-molecular-weight DEAE-dextran for large-scale experiments

With standard protocols for DNA infection, only a small percentage (about 4%) of monkey ells exposed to purified DNA of simian virus 40 (SV40) displays cytopathic effect or expresses viral T antigen. We have recently reported (Sompayrac and Danna, 1981) that by extending the time of exposure of BSC-1 cells to DNA in the presence of low concentrations of diethylaminoethyl (DEAE)-dextran, we can infect up to 50% of the cells. Because our protocol was devised for DEAE-dextran of mol. wt. 2 X 10(6), which is no longer commercially available, we have tested a low-molecular-weight polymer (mol. wt. 0.5 X 10(6) that can be purchased. Use of this reagent in our protocol resulted in marginally higher levels of infection than we found with the high-molecular-weight polymer. However, slightly more DNA (about 1.5 times as much) was needed to achieve saturation. With both reagents, the percentage of cells infected was proportional to time of exposure. Therefore, low-molecular-weight DEAE-dextran should be useful for large-scale DNA infections.

An SV40 mutant T antigen does not bind the SV40 viral origin

F8dl is an SV40 deletion mutant that lacks over 60% of the coding sequences for large T antigen and yet is able to immortalize early passage rat cells, to transform established cell lines, and to cause tumors in animals. We report here on the further characterization of this mutant and show that (a) transformation by F8dl is protein mediated but does not require the action of the SV40 small t antigen; (b) the F8dl T antigens have, or are associated with, an ATPase activity; (c) the 34-kDa mutant T antigen of F8dl is localized in nuclei and cell membranes of F8dl transformants and binds to double-stranded DNA; (d) the 20-25 kDa forms of the mutant T antigen are cytoplasmic; and (e) the F8dl T antigens do not bind with high affinity to the SV40 origin of viral DNA replication.

Chapter Ten Production of cellulases in plants for biomass conversion

Publisher Summary Plant biomass, the most abundant renewable resource on earth, is a potential source of fermentable sugars for the production of clean-burning, alternative transportation fuels such as ethanol. Bioconversion of plant biomass to fermentable glucose involves the enzymatic hydrolysis of cellulose, a major polysaccharide constituent of the plant cell wall. Enzymes from thermophilic organisms are particularly suited for this application because they are thermostable, resistant to protease, and tolerant toward other stresses. Commercially available microbial cellulases are prohibitively expensive for bioethanol processes. Manufacturing heterologous cellulases in crop-plant bioreactors could significantly reduce costs associated with enzyme production and could offer a potentially high-volume alternative to traditional methods. Because transgenic plants that produce large amounts of bacterial glucanases (up to 26% of total soluble leaf protein) exhibit normal growth and development, this approach offers much promise.