Expression of the p80 region of bovine viral diarrhea virus and identification of specific antibodies to this recombinant protein in bovine sera.

A 917-base pair segment of the bovine viral diarrhea virus (BVDV) genome encoding part of the p80 region was cloned into plasmid Gex-2T expression vector for expression as a fusion protein with glutathione-S-transferase (GST). When the p80 and GST sequences were in the same reading frames, the resulting GST-p80 fusion protein had a molecular mass of 58 kilodaltons (kDa) in SDS-PAGE. Extracts of control E. coli carrying only the vector plasmid (Gex-2T) did not contain this new 58-kDa protein band. Mouse monoclonal antibody specific to BVDV-p80 recognized this recombinant protein. Seventy cattle sera that had an SN titer (to TGAC isolate of cytopathic BVDV) greater than 1:8 reacted with this recombinant protein in Western blots. Of 28 cattle sera that had SN titers less than or equal to 1:8, only one serum tested positive on Western blots.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

UHRF2, a Ubiquitin E3 Ligase,Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.     

Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

Citric acid production by Aspergillus niger immobilized on polyurethane foam

Summary Citric acid was produced using Aspergillus niger immobilized on polyurethane foam in a bubble column reactor. Most of the adsorbed cells remained on the support and, as a result, high oxygen tension was maintained during the reactor operation. However, uncontrolled growth of the pellets made continuous reactor operation difficult. The citric acid productivity obtained from 15 vol.% foam particles containing immobilized cells was 0.135 g/l per hour. This productivity of immobilized cells was almost the same as that of free cells. The oxygen level dropped to half saturation in 5 days in the immobilized cell culture in contrast to 2 days in the free cell culture.     

Ultrastructure of poplar cortical cells during the transition from growing to wintering stages and vice versa

Summary Electron microscopic studies revealed that major cytological changes in the cortical cells of poplar (Populus euramericana cv. gelrica) began to occur in early September in conjunction with the metabolic transition from the growing to the wintering stage. During this transition, the cells became temporarily rich in endoplasmic reticulum, polysomes and vesicles. As the conspicuous formation of organelles progressed, the large vacuoles became smaller and filled with osmiophilic materials. Undefined organelles (protein-lipid bodies) also increased in number. From late October until March, organelles involved in protein synthesis were sparsely distributed in the cells, indicating that the number of these organelles is probably linked to the seasonal cycle of protein synthesis. In early February, after release from dormancy, fusion of vacuoles proceeded in the cells. The inclusion of organelles and a gradual decrease in the amount of osmiophilic materials in the vacuoles occurred at this stage. Subsequently, the structure of the cells continued to undergo changes to accommodate growth, which occurred in early May.     

Simian retrovirus-D serotype 1 (SRV-1) envelope glycoproteins gp70 and gp20: expression in yeast cells and identification of specific antibodies in sera from monkeys that recovered from SRV-1 infection.

The gp70 and transmembrane gp20 envelope proteins of simian retrovirus-D serotype 1 (SRV-1) were expressed in Saccharomyces cerevisiae as fusion proteins with human superoxide dismutase (SOD). Expression of the SOD-gp70 and SOD-gp20 sequences yielded fusion proteins of 52 and 29 kilodaltons, respectively. The yeast-expressed SRV-1 envelope proteins were used in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in the sera of rhesus macaques that recovered from SRV-1. Sera from 47 of 49 such monkeys tested positive for antibodies to the SOD-gp70 fusion protein, while 45 of 49 reacted positively to SOD-gp20. None of 26 SRV-1-nonexposed monkeys tested positive in either ELISA. Monkeys immunized with the recombinant SRV-1 gp20 and gp70 proteins made good ELISA and Western blot (immunoblot) antibodies to whole SRV-1. This antibody was not neutralizing in vitro, however.     

Mapping of the entomocidal fragment of Spodoptera -specific Bacillus thuringiensis toxin CryIC

 Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI poteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins. Received: 15 April 1996/Accepted: 10 July 1996