Intrinsic Bent DNA Sites in the Developmentally Amplified C3-22 Gene Promoter of Rhynchosciara americana (Diptera: Sciaridae)

The Rhynchosciara americana C3-22 gene is located in an amplified domain and is developmentally expressed. The aim of the present work was to identify intrinsically bent DNA sites in a segment containing the gene promoter and downstream sequence. The results indicated that this gene is flanked by intrinsically bent DNA sites. Three bent DNA sites (b^?3, b^?2, and b^?1) were localized in the promoter, and one was localized downstream of the gene (b⁺¹). These sites had helical parameters that confirmed the curved structure, as well as segments with left-handed superhelical writhe. In silico analysis of the promoters of four other insect genes, which encode secreted polypeptides, showed that they all had curved structures and similar helical parameters. Correlation with other results indicates that the detected intrinsically bent DNA sites that flank the C3-22 gene might be a consensus feature of the gene structure in the amplified domains.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Three distinct regulatory elements comprise the upstream promoter region of the nopaline synthase gene

Summary Fine deletion mutants were generated in the upstream control region of the nopaline synthase (nos) promoter to define the position and role of upstream regulatory elements. The results indicated that the 8 bp sequence (CAGAAACC) at -106/-113 and its inverted repeat (GGTTTCTG) at -140/-147 are important for promoter function. The downstream element appears more important than the upstream element since deletion of the former reduced promoter activity more significantly than deletion of the latter. Deletion of the element alone, however, did not abolish promoter function, whereas, deletion of the 10 bp potential Z-DNA-forming (Z) element located between the repeat elements nullified promoter activity. Therefore, it appears that the Z element is an essential upstream regulator and the repeated elements are upstream modulators of the nos promoter. These elements are functionally distinct since alteration of stereospecificity or insertion of short oligonucleotides between the elements did not significantly influence promoter activity. These regulatory elements were unable to function from 200 bp upstream of the CCAAT-TATA box region.     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.